U.S. patent number 9,675,697 [Application Number 14/204,076] was granted by the patent office on 2017-06-13 for bet bromodomain inhibitors and therapeutic methods using the same.
This patent grant is currently assigned to THE REGENTS OF THE UNIVERSITY OF MICHIGAN. The grantee listed for this patent is THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Irfan A. Asangani, Longchuan Bai, Arul Chinnaiyan, Hacer Karatas, Xiaoqin Li, Liu Liu, Ruijuan Luo, Donna McEachern, Jennifer Lynn Meagher, Xu Ran, Jeanne Stuckey, Duxin Sun, Shaomeng Wang, Chao-Yie Yang, Yujun Zhao, Bing Zhou.
United States Patent |
9,675,697 |
Wang , et al. |
June 13, 2017 |
BET bromodomain inhibitors and therapeutic methods using the
same
Abstract
Inhibitors of BET bromodomains and compositions containing the
same are disclosed. Methods of using the BET bromodomain inhibitors
in the treatment of diseases and conditions wherein inhibition of
BET bromodomain provides a benefit, like cancers, also are
disclosed.
Inventors: |
Wang; Shaomeng (Saline, MI),
Ran; Xu (Ann Arbor, MI), Zhao; Yujun (Ann Arbor, MI),
Yang; Chao-Yie (Ann Arbor, MI), Liu; Liu (Ann Arbor,
MI), Bai; Longchuan (Ann Arbor, MI), McEachern; Donna
(Ann Arbor, MI), Stuckey; Jeanne (Fenton, MI), Meagher;
Jennifer Lynn (Ann Arbor, MI), Sun; Duxin (Ann Arbor,
MI), Li; Xiaoqin (Ann Arbor, MI), Zhou; Bing (Ann
Arbor, MI), Karatas; Hacer (Ann Arbor, MI), Luo;
Ruijuan (Ann Arbor, MI), Chinnaiyan; Arul (Ann Arbor,
MI), Asangani; Irfan A. (Ann Arbor, MI) |
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN |
Ann Arbor |
MI |
US |
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Assignee: |
THE REGENTS OF THE UNIVERSITY OF
MICHIGAN (Ann Arbor, MI)
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Family
ID: |
51488537 |
Appl.
No.: |
14/204,076 |
Filed: |
March 11, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140256706 A1 |
Sep 11, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61936949 |
Feb 7, 2014 |
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61866126 |
Aug 15, 2013 |
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61775886 |
Mar 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
1/18 (20180101); A61P 13/02 (20180101); A61P
31/22 (20180101); A61P 37/06 (20180101); C07D
471/04 (20130101); A61P 43/00 (20180101); A61P
17/02 (20180101); A61P 11/00 (20180101); A61P
25/16 (20180101); A61P 3/10 (20180101); A61P
35/02 (20180101); A61P 37/08 (20180101); A61P
7/06 (20180101); A61P 29/00 (20180101); A61P
1/04 (20180101); A61P 17/14 (20180101); A61P
17/06 (20180101); A61P 31/14 (20180101); A61P
1/02 (20180101); A61P 13/12 (20180101); A61P
19/02 (20180101); A61P 7/02 (20180101); A61P
25/28 (20180101); A61P 35/00 (20180101); A61K
45/06 (20130101); A61P 1/16 (20180101); A61P
9/10 (20180101); C07D 487/04 (20130101); A61P
31/20 (20180101); A61P 37/02 (20180101); C07D
491/056 (20130101); A61P 11/02 (20180101); A61P
9/00 (20180101); A61P 25/00 (20180101); A61P
11/06 (20180101); A61P 31/04 (20180101); A61P
31/12 (20180101); A61P 7/00 (20180101); A61P
31/18 (20180101) |
Current International
Class: |
A61K
31/519 (20060101); C07D 491/056 (20060101); C07D
487/04 (20060101); A61K 45/06 (20060101); C07D
471/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0989131 |
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Mar 2000 |
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EP |
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WO-2008/092231 |
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Aug 2008 |
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WO |
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WO-2008/137816 |
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Nov 2008 |
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WO |
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WO-2009/075830 |
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Jun 2009 |
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WO |
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WO-2011/143669 |
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Nov 2011 |
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WO |
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WO-2012/075383 |
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Jun 2012 |
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WO |
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WO-2012/075456 |
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Jun 2012 |
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WO |
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WO-2012/174487 |
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Dec 2012 |
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WO |
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WO-2013/024104 |
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Feb 2013 |
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WO |
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WO-2013/027168 |
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Feb 2013 |
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WO |
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WO-2013/030150 |
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Mar 2013 |
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WO |
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WO-2013/033268 |
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Mar 2013 |
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WO |
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WO2013110198 |
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Aug 2013 |
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WO |
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WO-2014/134232 |
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Sep 2014 |
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WO |
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WO2015131005 |
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Sep 2015 |
|
WO |
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WO-2016/138332 |
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Sep 2016 |
|
WO |
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Other References
Garnier, J., et al. "BET Bromodomain inhibitors: a patent review."
Expert Opin. Ther. Patents. (2013), vol. 24(2), pp. 1-15. cited by
examiner .
Mayo Clinic. "Cancer". .COPYRGT. 2015. Available from: <
http://www.mayoclinic.org/diseases-conditions/cancer/basics/prevention/co-
n-20032378 >. cited by examiner .
Muller, S., et al. "Bromodomains as therapeutic targets." Expert
Reviews in Molecular Medicine. (Sep. 2011), vol. 13, e29, pp. 1-21.
cited by examiner .
Garnier, Jean-Marc, et al. "BET bromodomain inhibitors: a patent
review." Expert Opinion on Therapeutic Patents. .COPYRGT. Nov. 22,
2013. Available from: <
http://www.tandfonline.com/doi/pdf/10.1517/13543776.2014.859244
>. cited by examiner .
Belkina et al., BET domain co-regulators in obesity, inflammation
and cancer, Nat. Rev. Cancer, 12(7):465-77 (2012). cited by
applicant .
Filippakopoulos et al., Histone recognition and large-scale
structural analysis of the human bromodomain family, Cell,
149(1):214-31 (2012). cited by applicant .
Garnier et al., BET bromodomain inhibitors: a patent review, Exp.
Opin. Ther. Patents, 24:1-15 (2013). cited by applicant .
Haynes et al., The bromodomain: a conserved sequence found in
human, Drosophila and yeast proteins, Nucleic Acids Res.,
20(10):2603 (1992). cited by applicant .
Muller et al., Bromodomains as therapeutic targets, Expert Rev.
Mol. Med., 13:e29 (2011). cited by applicant .
Sanchez et al., The role of human bromodomains in chromatin biology
and gene transcription, Curr. Opin. Drug Discov. Devel.,
12(5):659-65 (2009). cited by applicant .
International Search Report in international application No.
PCT/US2014/022953, dated Jul. 24, 2014. cited by applicant .
Ferguson et al., Targeting Low-Druggability Bromodomains: Fragment
Based Screening and Inhibitor Design against the BAZ2B Bromodomain,
J. Med. Chem., 56:10183-10187 (2013). cited by applicant .
Extended European Search Report for European Patent Application No.
14779458.0, dated Jul. 13, 2016. cited by applicant.
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Primary Examiner: Jarrell; Noble
Assistant Examiner: Kenyon; John S
Attorney, Agent or Firm: Marshall, Gerstein & Borun
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional patent
application No. 61/775,886, filed Mar. 11, 2013; U.S. provisional
application No. 61/866,126, filed Aug. 15, 2013; and U.S.
provisional patent application No. 61/936,949, filed Feb. 7, 2014,
each incorporated herein by reference in its entirety.
Claims
What is claimed:
1. A compound having a structural formula (I): ##STR00609##
wherein: X is N(R.sup.a); Y.sup.1 and Y.sup.3, independently, are
CH or N; Y.sup.2 is CH, CR.sup.a, or N; Z is H, ##STR00610## halo,
or OH; A is ##STR00611## each unsubstituted or substituted; B is
aryl, CH(R.sup.a)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a)-heteroaryl, C.sub.3-10heterocycloalkyl, or
CH(R.sup.a)--C.sub.3-10heterocycloalkyl, each unsubstituted or
substituted; G is N, O, or S; L is null, H, or C(R.sup.d).sub.3;
R.sup.1 is H, halo, OH, OR.sup.a, or N(R.sup.a).sub.2; R.sup.a,
independently, is H, C.sub.1-3alkyl, or benzyl; R.sup.b,
independently, is C.sub.1-6alkyl, halo, aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, or CHO; n is an integer 0, 1,
2, or 3; and R.sup.c and R.sup.d, each independently, are hydrogen,
C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl; or a pharmaceutically
acceptable salt, hydrate, or solvate thereof.
2. The compound of claim 1, wherein ring A is: ##STR00612##
3. The compound of claim 1 or 2, wherein R.sup.1 is OCH.sub.3.
4. The compound of claim 1, wherein the ring system
##STR00613##
5. The compound of claim 1, wherein Z is ##STR00614##
6. The compound of claim 5, wherein the B ring, substituted or
unsubstituted, is selected from the group consisting of:
##STR00615## ##STR00616##
7. The compound of claim 6, wherein the B ring is substituted with
one to three of methyl, phenyl, fluoro, pyridinyl, chloro,
isopropyl, cyclopropyl, or ethyl.
8. The compound of claim 1 selected from the group consisting of:
##STR00617## ##STR00618## ##STR00619## ##STR00620## ##STR00621##
##STR00622## ##STR00623## ##STR00624## ##STR00625## ##STR00626##
##STR00627## or a pharmaceutically acceptable salt, hydrate, or
solvate thereof.
9. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier or vehicle.
10. A method of reducing or ameliorating breast cancer, leukemia,
or prostate cancer, the method comprising administering a
therapeutically effective amount of a compound of claim 1 to an
individual in need thereof.
11. The method of claim 10 further comprising administering a
therapeutically effective amount of a second therapeutic agent
useful in the treatment of cancer.
12. The method of claim 11, wherein the compound of claim 1 and the
second therapeutic agent are administered simultaneously.
13. The method of claim 11, wherein the compound of claim 1 and the
second therapeutic agent are administered separately.
14. The method of claim 11, wherein the second therapeutic agent is
one or more of surgery, a chemotherapeutic agent, or radiation.
15. The method of claim 11, wherein the compound of claim 1 and the
second therapeutic agent are administered from a single
composition.
16. The method of claim 11, wherein the compound of claim 1 and the
second therapeutic agent are administered from separate
compositions.
17. The method of claim 13, wherein the compound of claim 1 is
administered prior to the second therapeutic agent.
18. The method of claim 13, wherein the compound of claim 1 is
administered after the second therapeutic agent.
19. The compound of claim 8 which is: ##STR00628## or a
pharmaceutically acceptable salt, hydrate, or solvate thereof.
20. A method of reducing or ameliorating breast cancer, leukemia,
or prostate cancer, the method comprising administering a
therapeutically effective amount of a compound of claim 8 to an
individual in need thereof.
21. A method of reducing or ameliorating breast cancer, leukemia,
or prostate cancer, the method comprising administering a
therapeutically effective amount of a compound of claim 19 to an
individual in need thereof.
Description
FIELD OF THE INVENTION
The present invention relates to BET bromodomain inhibitors and to
therapeutic methods of treating conditions and diseases wherein
inhibition of BET bromodomains provides a benefit.
BACKGROUND OF THE INVENTION
The genomes of eukaryotic organisms are highly organized within the
nucleus of the cell. The long strands of duplex DNA are wrapped
around an octamer of histone proteins (usually comprising two
copies of histones H2A, H2B, H3, and H4) to form a nucleosome,
which then is further compressed to form a highly condensed
chromatin structure. A range of different condensation states are
possible, and the tightness of this structure varies during the
cell cycle. The chromatin structure plays a critical role in
regulating gene transcription, which cannot occur efficiently from
highly condensed chromatin. The chromatin structure is controlled
by a series of post translational modifications to histone
proteins, notably histones H3 and H4. These modifications include
acetylation, methylation, phosphorylation, ubiquitinylation, and
SUMOylation.
Histone acetylation usually is associated with the activation of
gene transcription, as the modification loosens the interaction of
the DNA and the histone octomer by changing the electrostatics. In
addition to this physical change, specific proteins bind to
acetylated lysine residues within histones to read the epigenetic
code. Bromodomains are small (about 110 amino acid) distinct
domains within proteins that bind to acetylated lysine resides
commonly, but not exclusively, in the context of histones. There is
a family of about 50 proteins known to contain bromodomains, which
have a range of functions within the cell.
The BET family of bromodomain-containing proteins includes four
proteins, i.e., BRD2, BRD3, BRD4, and BRD-t, which contain tandem
bromodomains capable of binding to two acetylated lysine residues
in close proximity, thereby increasing the specificity of the
interaction. BRD2 and BRD3 associate with histones along actively
transcribed genes and may be involved in facilitating
transcriptional elongation, while BRD4 may be involved in the
recruitment of the pTEF-.beta. complex to inducible genes,
resulting in phosphorylation of RNA polymerase and increased
transcriptional output. BRD4 or BRD3 also may fuse with NUT
(nuclear protein in testis) forming novel fusion oncogenes,
BRD4-NUT or BRD3-NUT, in a highly malignant form of epithelial
neoplasia. Data suggests that BRD-NUT fusion proteins contribute to
carcinogenesis. BRD-t is uniquely expressed in the testes and
ovary. All family members have been reported to have some function
in controlling or executing aspects of the cell cycle, and have
been shown to remain in complex with chromosomes during cell
division, which suggests a role in the maintenance of epigenetic
memory. In addition, some viruses make use of these proteins to
tether their genomes to the host cell chromatin as part of the
process of viral replication.
A discussion of BET proteins can be found in WO 2012/075456, WO
2012/075383, and WO 2011/054864, each designating the U.S. and each
incorporated herein by reference in its entirety. A discussion of
BET bromodomain inhibitors, e.g., 1-BET-151 and 1-BET-762, can be
found in Delmore et al., Cell 146:904-917 (2011) and Seal et al.,
Bioorg. Med. Chem. Lett. 22:2968-2972 (2012).
Despite research directed to BET bromodomains and BET bromodomain
inhibitors, the design of potent, non-peptide inhibitors of BET
bromodomains remains a significant challenge in modern drug
discovery. Accordingly, a need still exists in the art for BET
bromodomain inhibitors having physical and pharmacological
properties that permit use of the inhibitors in therapeutic
applications. The present invention provides compounds designed to
bind to BET bromodomains and inhibit BET bromodomain activity.
SUMMARY OF THE INVENTION
The present invention is directed to inhibitors of BET
bromodomains, to compositions comprising the inhibitors, and to
methods of using the inhibitors in a therapeutic treatment of
conditions and diseases wherein inhibition of BET bromodomain
activity provides a benefit.
In one aspect, the present invention is directed to compounds
having a structural formula (I):
##STR00001##
wherein:
X is N(R.sup.a), O, or S;
Y.sup.1 and Y.sup.3, independently, are CH or N;
Y.sup.2 is CH, CR.sup.a, N, or null;
Z is H,
##STR00002## halo, OH, or null;
A is an unsubstituted or substituted 5-membered heterocyclic
ring;
B is aryl, CH(R.sup.a)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a)-heteroaryl, C.sub.3-10heterocycloalkyl, or
CH(R.sup.a)--C.sub.3-10heterocycloalkyl, each unsubstituted or
substituted;
G is N, O, or S;
L is null, H, or C(R.sup.d).sub.3;
R.sup.1 is H, halo, OH, OR.sup.a, or N(R.sup.a).sub.2;
R.sup.a, independently, is H, C.sub.1-3alkyl, or benzyl;
R.sup.b, independently, is C.sub.1-6alkyl, halo, aryl,
unsubstituted or substituted CH.sub.2-aryl, unsubstituted or
substituted C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, or CHO;
n is an integer 0, 1, 2, or 3;
R.sup.c and R.sup.d, each independently, are hydrogen,
C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl;
or a pharmaceutically acceptable salt, hydrate, or solvate
thereof.
In another aspect, the present invention is directed to compounds
having a structural formula (I):
##STR00003##
wherein:
X is N(R.sup.a1), O, or S;
Y.sup.1 and Y.sup.3, independently, are CH or N;
Y.sup.2 is CR.sup.2, N, or null;
Z is H,
##STR00004## halo, or OH;
A is an unsubstituted or substituted 5-membered heterocyclic
ring;
B is aryl, CH(R.sup.a2)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a2)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a2)-heteroaryl, C.sub.3-10heterocycloalkyl,
CH(R.sup.a2)--C.sub.3-10heterocycloalkyl,
##STR00005## each unsubstituted or substituted;
G is N, O, or S;
L is null, H, or C(R.sup.d).sub.3;
R.sup.1 is H, halo, OH, OR.sup.a3, R.sup.a3, or
N(R.sup.a3).sub.2;
R.sup.a1, R.sup.a2, R.sup.a3, R.sup.a4, and R.sup.a5 each
independently, is H, C.sub.1-3alkyl, phenyl, or benzyl;
R.sup.2, independently, is H, C.sub.1-3alkyl,
(CH.sub.2).sub.1-3C.sub.4-7heterocycloalkyl,
C.sub.4-7heterocycloalkyl, CO.sub.2H, CO.sub.2(C.sub.1-3alkyl),
NH.sub.2, NH(C.sub.1-3alkyl), N(C.sub.1-3alkyl).sub.2,
(CH.sub.2).sub.1-3NMe.sub.2, (CH.sub.2).sub.1-3OH, C(Me).sub.2OH,
CH(Me)OH, C(Me.sub.2)NH.sub.2, phenyl, benzyl, C(.dbd.O)OR.sup.a4,
C(.dbd.O)N(R.sup.a4).sub.2, C(.dbd.O)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl, C(.dbd.O)-unsubstituted or substituted
hydroxyC.sub.3-10heterocycloalkyl,
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3unsubstituted or substituted
C.sub.3-10heterocycloalkyl, C(.dbd.O)N(R.sup.a1)-unsubstituted or
substituted C.sub.3-10heterocycloalkyl,
C(.dbd.O)N(R.sup.a1)-hydroxycycloalkyl,
C(.dbd.O)N(R.sup.a1)--C.sub.1-6hydroxyalkyl,
##STR00006##
R.sup.b, independently, is: C.sub.1-6alkyl, C.sub.1-6hydroxyalkyl,
halo, aryl, unsubstituted or substituted CH.sub.2-aryl,
unsubstituted or substituted C.sub.3-10cycloalkyl, unsubstituted or
substituted CH.sub.2--C.sub.3-10cycloalkyl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted
CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, CF.sub.3, CN, OR.sup.a5
N(R.sup.a5).sub.2, N(R.sup.a1)C.sub.1-6 hydroxyalkyl
N(R.sup.a1)C(.dbd.O)(C.sub.1-6alkyl),
N(R.sup.a1)C(.dbd.O)(CH.sub.2).sub.1-3-unsubstituted or substituted
C.sub.3-10heterocycloalkyl,
N(R.sup.a1)C(.dbd.O)(CH.sub.2).sub.1-3-hydroxyC.sub.3-10heterocycloalkyl,
NH(CH.sub.2).sub.2-3CO.sub.2H,
NH(CH.sub.2).sub.2-3C(.dbd.O)N(R.sup.a5).sub.2,
N(R.sup.a1)C(.dbd.O)(CH.sub.2).sub.1-3N(H)-unsubstituted or
substituted C.sub.3-10heterocycloalkyl,
N(R.sup.a1)C(.dbd.O)(CH.sub.2).sub.1-3-unsubstituted or substituted
C.sub.3-10heterocycloalkyl,
N(R.sup.a1)C(.dbd.O)(CH.sub.2).sub.1-3N(H)--C.sub.1-6hydroxyalkyl,
N(R.sup.a1)C(.dbd.O)N(R.sup.a2).sub.2,
N(R.sup.a1)C(.dbd.O)N(R.sup.a2)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl N(R.sup.a1)C(.dbd.O)N(R.sup.a2).sub.2,
N(R.sup.a1)C(.dbd.O)N(R.sup.a2)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl
NH(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
NH(CH.sub.2).sub.2-3--C.sub.3-10heterocycloalkyl,
N[(CH.sub.2).sub.2-3--C.sub.3-10heterocycloalkyl].sub.2,
O(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
O(CH.sub.2).sub.2-3--C.sub.3-10heterocycloalkyl,
C(.dbd.O)N(R.sup.a5).sub.2
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3-unsubstituted or substituted
C.sub.3-10heterocycloalkyl, C(.dbd.O)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl,
N(R.sup.a1)C(.dbd.O)-hydroxyC.sub.3-10heterocycloalkyl,
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3--N(H)C(.dbd.O)NH.sub.2,
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2
C(.dbd.O)N(R.sup.a1)(CH.sub.2).sub.2-3--CO.sub.2R.sup.a1,
C(.dbd.O)N(R.sup.a1)-alkyl,
C(.dbd.O)N(R.sup.a1)--C.sub.1-6hydroxyalkyl,
C(.dbd.O)N(R.sup.a1)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl,
C(.dbd.O)N(R.sup.a1)--C.sub.1-6hydroxyalkyl,
C(.dbd.O)N(R.sup.a1)CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3,
C(.dbd.O)N(R.sup.a1)CH.sub.2CH.sub.2SO.sub.2Me, CO.sub.2R.sup.a1,
C(R.sup.a1).sub.2CO.sub.2R.sup.a2,
C(R.sup.a1).sub.2C(.dbd.O)N(R.sup.a5).sub.2,
C(R.sup.a1).sub.2C(.dbd.O)N(R.sup.a2)-unsubstituted or substituted
C.sub.3-10heterocycloalkyl, C(R.sup.a1).sub.2CN,
##STR00007## oxo(.dbd.O), or CHO;
n is an integer 0, 1, 2, or 3;
m is an integer 0, 1, 2, or 3;
R.sup.c and R.sup.d, each independently, are hydrogen,
C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, hydroxycycloalkyl, or unsubstituted or
substituted CH.sub.2--C.sub.3-10heterocycloalkyl, or
R.sup.c and R.sup.d taken together form an unsubstituted or
substituted C.sub.3-10heterocycloalkyl or
hydroxyC.sub.3-10heterocycloalkyl;
Q.sup.- is a pharmaceutically acceptable anion;
or a pharmaceutically acceptable salt, hydrate, or solvate
thereof.
In one embodiment, the present invention provides a method of
treating a condition or disease by administering a therapeutically
effective amount of a compound of structural formula (I) to an
individual in need thereof. The disease or condition of interest is
treatable by inhibition of BET bromodomains, for example, a cancer,
a chronic autoimmune disorder, an inflammatory condition, a
proliferative disorder, sepsis, or a viral infection.
Another embodiment of the present invention is to provide a
composition comprising (a) a BET bromodomain inhibitor of
structural formula (I) and (b) an excipient and/or pharmaceutically
acceptable carrier useful in treating diseases or conditions
wherein inhibition of BET bromodomains provides a benefit.
Another embodiment of the present invention is to utilize a
composition comprising a compound of structural formula (I) and a
second therapeutically active agent in a method of treating an
individual for a disease or condition wherein inhibition of BET
bromodomains, e.g., BRD2, BRD3, BRD4, BRD-t, or an isoform or
mutant thereof, provides a benefit.
In a further embodiment, the invention provides for use of a
composition comprising a BET bromodomain inhibitor of structural
formula (I) and an optional second therapeutic agent for the
manufacture of a medicament for treating a disease or condition of
interest, e.g., a cancer.
Still another embodiment of the present invention is to provide a
kit for human pharmaceutical use comprising (a) a container, (b1) a
packaged composition comprising a BET bromodomain inhibitor of
structural formula (I), and, optionally, (b2) a packaged
composition comprising a second therapeutic agent useful in the
treatment of a disease or condition of interest, and (c) a package
insert containing directions for use of the composition or
compositions, administered simultaneously or sequentially, in the
treatment of the disease or condition.
A BET bromodomain inhibitor of structural formula (I) and the
second therapeutic agent can be administered together as a
single-unit dose or separately as multi-unit doses, wherein the BET
bromodomain inhibitor of structural formula (I) is administered
before the second therapeutic agent or vice versa. It is envisioned
that one or more dose of a BET bromodomain inhibitor of structural
formula (I) and/or one or more dose of a second therapeutic agent
can be administered.
In one embodiment, a BET bromodomain inhibitor of structural
formula (I) and a second therapeutic agent are administered
simultaneously. In related embodiments, a BET bromodomain inhibitor
of structural formula (I) and a second therapeutic agent are
administered from a single composition or from separate
compositions. In a further embodiment, the BET bromodomain
inhibitor of structural formula (I) and second therapeutic agent
are administered sequentially. A BET bromodomain inhibitor of
structural formula (I), as used in the present invention, can be
administered in an amount of about 0.005 to about 500 milligrams
per dose, about 0.05 to about 250 milligrams per dose, or about 0.5
to about 100 milligrams per dose.
These and other embodiments and features of the present invention
will become apparent from the following detailed description of the
preferred embodiments.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a line graph showing in vivo antitumor activity of Cpd.
No. 73 in the MDA-MB-436 breast cancer xenograft model in SCID
mice. Tumors were grown s.c. to an average size of 100 mm.sup.3,
and Cpd. No. 73 was administered orally at the indicated dose and
schedule. Vehicle Control (PEG200) was given twice daily.
FIG. 2 is a line graph showing the animal weight following
administration of Cpd. No. 73 in in MDA-MB-436 tumor-bearing SCID
mice. Cpd. No. 73 was administered orally at the indicated dose and
schedule. Vehicle Control (PEG200) was given twice daily.
FIG. 3 is a line graph showing in vivo antitumor activity of Cpd.
No. 73 in the MDA-MB-231 breast cancer xenograft model in mice.
Cpd. No. 73 was administered with daily oral, dosing via oral
gavage with either 20 or 40 mg/kg for 12 days. Each group had eight
mice and each mouse bearing one tumor.
FIG. 4 is an illustration showing western blot analysis of in vivo
upregulation of p21 and by BET inhibitors in MV-4;11 xenograft
tumors in SCID mice. Compounds were dosed orally at 100 mg/kg for
up to 72 hours. Resected xenograft tumor tissues were grinded into
powder in liquid nitrogen and lysed in lysis buffer [1% CHAPS, 150
mM NaCl, 20 mM Tris-HCl, 1 mM. EDTA, 1 mM EGTA, and COMPLETE
proteinase inhibitor (Roche)] for 2 freeze-thaw (-80.degree. C. to
room temperature) cycles then another 30 minutes on ice. Protein
concentrations were determined using the Bio-Rad Protein Assay Dye
reagent. Whole tumor lysates (20 .mu.g) were separated on a 4-20%
Novex gels (Invitrogen). The separated proteins were transferred to
a PVDF membrane (BIO-RAD) and the PVDF membrane was then blotted
with 5% Blotting-Grade Blocker (BIO-RAD) for 1 hour at room
temperature. The primary antibodies used were: p21Waf1/Cip1 (12D1)
Rabbit mAb [Cell Signaling technology (CST), Cat#2947] and PARP
(46D11) Rabbit mAb [CST #9532]. The secondary antibody used was
horseradish peroxidase conjugated goat anti-rabbit (Thermo
Scientific Cat#31460). The BIO-RAD Clarity Western ECL Substrates
(BIO-RAD) and HyBlot CL film (Denville) were used for signal
development and detection using a SRX-101A tabletop processor
(Konica Minolta).
FIG. 5 is a line graph showing in vivo antitumor activity of BET
inhibitors in the MV4;11 acute myeloid leukemia (AML) xenograft
model in SCID mice. Tumors were grown s.c. to an average size of
150 mm.sup.3, and Cpd. No. 73 was administered orally at the
indicated dose and schedule.
FIG. 6 is a line graph showing the animal weight following
administration of BET inhibitors in MV4;11 tumor-bearing SCID
mice.
FIG. 7 is an illustration showing two western blot analyses of p21
activation and apoptosis induced by BET inhibitors in MV-4;11
xenograft tumors in SCID mice. MV4-11 xenografts were treated with
drugs at 50 mg/kg for 6 and 24 hours. Resected xenograft tumor
tissues were grinded into powder in liquid nitrogen and lysed in
lysis buffer [1% CHAPS, 150 mM NaCl, 20 mM Tris-HCl, 1 mM. EDTA, 1
mM EGTA, and COMPLETE proteinase inhibitor (Roche)] for 2
freeze-thaw (-80.degree. C. to room temperature) cycles then
another 30 minutes on ice. Protein concentrations were determined
using the Bio-Rad Protein Assay Dye reagent. Whole tumor lysates
(20 .mu.g) were separated on a 4-20% Novex gels (Invitrogen). The
separated proteins were transferred to a PVDF membrane (BIO-RAD)
and the PVDF membrane was then blotted with 5% Blotting-Grade
Blocker (BIO-RAD) for 1 hour at room temperature. The primary
antibodies used were: p21Waf1/Cip1 (12D1) Rabbit mAb [Cell
Signaling technology (CST), Cat#2947] and PARP (46D11) Rabbit mAb
[CST #9532]. The secondary antibody used was horseradish peroxidase
conjugated goat anti-rabbit (Thermo Scientific Cat#31460). The
BIO-RAD Clarity Western ECL Substrates (BIO-RAD) and HyBlot CL film
(Denville) were used for signal development and detection using a
SRX-101A tabletop processor (Konica Minolta).
FIG. 8 is a bar graph showing AR target (PSA, ERG) and MYC
(positive control) expression as measured by QRT-PCR analysis in
VCaP cells treated with DMSO or 0.5 .mu.M of the indicated BET
Bromodomain inhibitor for 24 hours.
FIG. 9 is a bar graph showing MYC mRNA expression as measured by
QRT-PCR analysis in AR negative DU145 cells treated with DMSO or
0.5 .mu.M of the indicated BET Bromodomain inhibitor for 24
hours.
FIG. 10 is an illustration showing BRD4 de-recruitment from KLK3
(PSA) loci by BET inhibitors as measured by ChIP-seq. Starved VCaP
cells were treated with 0.5 uM JQ1 or Cpd. No. 68for 5 hrs prior to
12 hrs DHT stimulation, followed by BRD4 ChIP-seq. Anti-androgens
MDV3100 (10 uM) and Bicaluatmide (25 uM) were used for comparative
purpose. Figure depicts genome browser view of BRD4 binding events
on AR regulated KLK3 loci. The y-axis denotes reads per million per
base pair (rpm/bp). The x-axis denotes the genomic position with a
scale bar on top right.
FIG. 11 is an illustration showing immunoblot analysis of PSA, ERG,
and MYC proteins in starved VCaP cells pre-treated with vehicle,
MDV3100 (10 .mu.M), JQ1 (0.5 uM) or CD-225 alone or in combination
as indicated for 4 hrs followed by DHT (10 nM) for 20 hrs. GAPDH
was used as loading control.
FIG. 12 is a line graph showing in vivo anti-tumor activity of Cpd.
No. 73 in a VCaP prostate cancer mouse xenograph model. VCaP cells
were implanted subcutaneously in mice and grown until tumors
reached the size of approximately 100 mm.sup.3. Xenografted mice
were randomized and then received vehicle or 40 mg/kg Cpd. No. 73
by oral gavage for 5 days/week. Mean tumor volume.+-.SEM is
shown.
FIG. 13 is a line graph showing in vivo anti-tumor activity of Cpd.
No. 73 in a VCaP prostate cancer mouse xenograph model. VCaP cells
were implanted subcutaneously in mice and grown until tumors
reached the size of approximately 100 mm.sup.3. Xenografted mice
were randomized and then received vehicle or 40 mg/kg Cpd. No. 73
by oral gavage for 13 days. Treatment was stopped from day 13
onwards and the animals were observed for tumor growth. Mean tumor
volume.+-.SEM is shown.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is described in connection with preferred
embodiments. However, it should be appreciated that the invention
is not limited to the disclosed embodiments. It is understood that,
given the description of the embodiments of the invention herein,
various modifications can be made by a person skilled in the art.
Such modifications are encompassed by the claims below.
The term "BET bromodomain" as used herein means one or more of
BRD2, BRD3, BRD4, and BRD-t.
The term "a disease or condition wherein inhibition of BET
bromodomains provides a benefit" pertains to a condition in which
at least one of BRD2, BRD3, BRD4, and BRD-t, and/or an action of at
least one of BRD2, BRD3, BRD4, and BRD-t, is important or
necessary, e.g., for the onset, progress, expression of that
disease or condition, or a disease or a condition which is known to
be treated by a BET bromodomain inhibitor. Examples of such
conditions include, but are not limited to, a cancer, a chronic
autoimmune disease, an inflammatory disease, a proliferative
disease, sepsis, and a viral infection. One of ordinary skill in
the art is readily able to determine whether a compound treats a
disease or condition mediated by a BET bromodomain for any
particular cell type, for example, by assays which conveniently can
be used to assess the activity of particular compounds.
The term "second therapeutic agent" refers to a therapeutic agent
different from a BET bromodomain inhibitor of structural formula
(I) and that is known to treat the disease or condition of
interest. For example when a cancer is the disease or condition of
interest, the second therapeutic agent can be a known
chemotherapeutic drug, like taxol, or radiation, for example.
The term "disease" or "condition" denotes disturbances and/or
anomalies that as a rule are regarded as being pathological
conditions or functions, and that can manifest themselves in the
form of particular signs, symptoms, and/or malfunctions. As
demonstrated below, a compound of structural formula (I) is a
potent inhibitor of BET bromodomains and can be used in treating
diseases and conditions wherein inhibition of BET bromodomains
provides a benefit.
As used herein, the terms "treat," "treating," "treatment," and the
like refer to eliminating, reducing, or ameliorating a disease or
condition, and/or symptoms associated therewith. Although not
precluded, treating a disease or condition does not require that
the disease, condition, or symptoms associated therewith be
completely eliminated. As used herein, the terms "treat,"
"treating," "treatment," and the like may include "prophylactic
treatment," which refers to reducing the probability of
redeveloping a disease or condition, or of a recurrence of a
previously-controlled disease or condition, in a subject who does
not have, but is at risk of or is susceptible to, redeveloping a
disease or condition or a recurrence of the disease or condition.
The term "treat" and synonyms contemplate administering a
therapeutically effective amount of a compound of the invention to
an individual in need of such treatment.
Within the meaning of the invention, "treatment" also includes
relapse prophylaxis or phase prophylaxis, as well as the treatment
of acute or chronic signs, symptoms and/or malfunctions. The
treatment can be orientated symptomatically, for example, to
suppress symptoms. It can be effected over a short period, be
oriented over a medium term, or can be a long-term treatment, for
example within the context of a maintenance therapy.
The term "therapeutically effective amount" or "effective dose" as
used herein refers to an amount of the active ingredient(s) that
is(are) sufficient, when administered by a method of the invention,
to efficaciously deliver the active ingredient(s) for the treatment
of condition or disease of interest to an individual in need
thereof. In the case of a cancer or other proliferation disorder,
the therapeutically effective amount of the agent may reduce (i.e.,
retard to some extent and preferably stop) unwanted cellular
proliferation; reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., retard to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
retard to some extent and preferably stop) tumor metastasis;
inhibit, to some extent, tumor growth; reduce BET bromodomain
signaling in the target cells; and/or relieve, to some extent, one
or more of the symptoms associated with the cancer. To the extent
the administered compound or composition prevents growth and/or
kills existing cancer cells, it may be cytostatic and/or
cytotoxic.
The term "container" means any receptacle and closure therefor
suitable for storing, shipping, dispensing, and/or handling a
pharmaceutical product.
The term "insert" means information accompanying a pharmaceutical
product that provides a description of how to administer the
product, along with the safety and efficacy data required to allow
the physician, pharmacist, and patient to make an informed decision
regarding use of the product. The package insert generally is
regarded as the "label" for a pharmaceutical product.
"Concurrent administration," "administered in combination,"
"simultaneous administration," and similar phrases mean that two or
more agents are administered concurrently to the subject being
treated. By "concurrently," it is meant that each agent is
administered either simultaneously or sequentially in any order at
different points in time. However, if not administered
simultaneously, it is meant that they are administered to an
individual in a sequence and sufficiently close in time so as to
provide the desired therapeutic effect and can act in concert. For
example, a BET bromodomain inhibitor of structural formula (I) can
be administered at the same time or sequentially in any order at
different points in time as a second therapeutic agent. A present
BET bromodomain inhibitor and the second therapeutic agent can be
administered separately, in any appropriate form and by any
suitable route. When a present BET bromodomain inhibitor and the
second therapeutic agent are not administered concurrently, it is
understood that they can be administered in any order to a subject
in need thereof. For example, a present BET bromodomain inhibitor
can be administered prior to (e.g., 5 minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second therapeutic agent treatment modality
(e.g., radiotherapy), to an individual in need thereof. In various
embodiments, a BET bromodomain inhibitor of structural formula (I)
and the second therapeutic agent are administered 1 minute apart,
10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour
apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours
to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours
apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours
to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours
apart, 11 hours to 12 hours apart, no more than 24 hours apart or
no more than 48 hours apart. In one embodiment, the components of
the combination therapies are administered at 1 minute to 24 hours
apart.
The use of the terms "a", "an", "the", and similar referents in the
context of describing the invention (especially in the context of
the claims) are to be construed to cover both the singular and the
plural, unless otherwise indicated. Recitation of ranges of values
herein merely are intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. The use of any and all examples, or exemplary
language (e.g., "such as") provided herein, is intended to better
illustrate the invention and is not a limitation on the scope of
the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Research has established that targeting BET bromodomains using
small molecule inhibitors is a viable cancer therapeutic strategy.
The prior discovery of BET bromodomain inhibitors and early data
have demonstrated that non-peptide, small molecule inhibitors of
BET bromodomains have great therapeutic potential for the treatment
of many diseases and conditions in which BET bromodomains have a
role.
The present invention is directed to a new class of potent and
specific inhibitors of BET bromodomains. The present compounds bind
to BET bromodomains and function as potent antagonists of BET
bromodomains. BET bromodomain inhibitors of the present invention
therefore are useful in the treatment of a variety of diseases and
conditions, including cancers and autoimmune diseases, in subjects
in need of such treatment. Also provided are methods of treating a
subject having unwanted proliferative cells comprising
administering a therapeutically effective amount of a present
compound to a subject in need of such treatment. Also provided are
methods of preventing the proliferation of unwanted proliferating
cells, such as cancers, in a subject comprising the step of
administering a therapeutically effective amount of a compound of
structural formula (I) to a subject at risk of developing a
condition characterized by unwanted proliferating cells. In some
embodiments, the compounds of structural formula (I) reduce the
proliferation of unwanted cells by inducing apoptosis in those
cells.
In one aspect, the present invention is drawn to the following
particular embodiments:
Embodiment I
A compound having a structural formula (I):
##STR00008## wherein
X is N(R.sup.a), O, or S;
Y.sup.1 and Y.sup.3, independently, are CH or N;
Y.sup.2 is CH, CR.sup.a, N, or null;
Z is H,
##STR00009## halo, OH, or null;
A is an unsubstituted or substituted 5-membered heterocyclic
ring;
B is aryl, CH(R.sup.a)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a)-heteroaryl, C.sub.3-10heterocycloalkyl, or
CH(R.sup.a)--C.sub.3-10heterocycloalkyl, each unsubstituted or
substituted;
G is N, O, or S;
L is null, H, or C(R.sup.d).sub.3;
R.sup.1 is H, halo, OH, OR.sup.a, or N(R.sup.a).sub.2;
R.sup.a, independently, is H, C.sub.1-3alkyl, or benzyl;
R.sup.b, independently, is C.sub.1-6alkyl, halo, aryl,
unsubstituted or substituted CH.sub.2-aryl, unsubstituted or
substituted C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, or CHO;
n is an integer 0, 1, 2, or 3;
R.sup.c and R.sup.d, each independently, are hydrogen,
C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl;
or a pharmaceutically acceptable salt, hydrate, or solvate
thereof.
Embodiment II
The compound of Embodiment I, wherein ring A is an optionally
substituted heteroaryl ring.
Embodiment III
The compound of Embodiment I, wherein ring A is optionally
substituted:
##STR00010##
Embodiment IV
The compound of Embodiment I, wherein ring A is optionally
substituted
##STR00011##
Embodiment V
The compound of Embodiment I wherein ring A is
##STR00012##
Embodiment VI
The compound of any of Embodiments I through V wherein R.sup.1 is H
or --OCH.sub.3.
Embodiment VII
The compound of any of Embodiments I through VI wherein the ring
system
##STR00013##
Embodiment VIII
The compound of Embodiments I through VII wherein Z is
##STR00014##
Embodiment IX
The compound of Embodiment VIII wherein the B ring, substituted or
unsubstituted, is selected from the group consisting of
##STR00015## ##STR00016##
Embodiment X
The compound of Embodiment IX, wherein the B ring is substituted
with one to three of methyl, phenyl, fluoro, pyridinyl, chloro,
isopropyl, cyclopropyl, and ethyl.
Embodiment XI
A compound having the structure set forth as follows:
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026##
Embodiment XII
A composition comprising (a) compound of Embodiment I, (b) a second
therapeutic agent useful in the treatment of a disease or condition
wherein inhibition of BET bromodomain protein provides a benefit,
and (c) an optional excipient and/or pharmaceutically acceptable
carrier.
Embodiment XIII
The composition of Embodiment XII, wherein the second therapeutic
agent comprises a chemotherapeutic agent useful in the treatment of
cancer.
Embodiment XIV
A pharmaceutical composition comprising a compound of Embodiment I
and a pharmaceutically acceptable carrier or vehicle.
Embodiment XV
A method of treating a disease or condition wherein inhibition of
BET bromodomain protein provides a benefit comprising administering
a therapeutically effective amount of a compound of Embodiment I to
an individual in need thereof.
Embodiment XVI
The method of Embodiment XV further comprising administering a
therapeutically effective amount of a second therapeutic agent
useful in the treatment of the disease or condition.
Embodiment XVII
The method of Embodiment XVI, wherein the compound of Embodiment I
and the second therapeutic agent are administered
simultaneously.
Embodiment XVIII
The method of Embodiment XVI, wherein the compound of Embodiment I
and the second therapeutic agent are administered separately.
Embodiment XIX
The method of Embodiment XV, wherein the disease or condition is a
cancer, a chronic autoimmune disorder, an inflammatory condition, a
proliferative disorder, sepsis, or a viral infection.
Embodiment XX
The method of Embodiment XVI, wherein the disease is a cancer and
the second therapeutic agent is one or more of surgery, a
chemotherapeutic agent, and radiation.
Embodiment XXI
The method of Embodiment XVI, wherein the disease is a cancer and
the second therapeutic agent is selected from the agents disclosed
in the specification.
Embodiment XXII
The method of Embodiment XVI, wherein the disease is a cancer and
the second therapeutic agent comprises radiation disclosed in the
specification.
Embodiment XXIII
The method of Embodiment XIX, wherein the cancer is selected from a
cancer disclosed in the specification.
Embodiment XXIV
The method of Embodiment XVI, wherein the compound of Embodiment I
and the second therapeutic agent are administered from a single
composition.
Embodiment XXV
The method of Embodiment XVI, wherein the compound of Embodiment I
and the second therapeutic agent are administered from separate
compositions.
Embodiment XXVI
The method of Embodiment XVIII, wherein the compound of Embodiment
I is administered prior to the second therapeutic agent.
Embodiment XXVII
The method of Embodiment XVIII, wherein the compound of Embodiment
I is administered after the second therapeutic agent.
Embodiment XXVIII
The method of Embodiment XIX, wherein the proliferative disorder is
selected from a disorder disclosed in the specification.
Embodiment XXIX
The method of Embodiment XIX, wherein the autoimmune disorder or
inflammatory is disorder is selected from a disorder disclosed in
the specification.
Embodiment XXX
The method of Embodiment XIX, wherein the viral infection is
selected from the infection disclosed in the specification
Embodiment XXXI
A method of inhibiting activity of a BET bromodomain protein, or a
mutant thereof, in a biological sample comprising contacting the
biological sample with a compound according to any one of
Embodiments I-XI or a composition according to Embodiment XIV.
Embodiment XXXII
The use of a compound according to any one of Embodiments I-XI in
the manufacture of a medicament for the treatment of a disease or a
condition for which a BET bromodomain inhibitor is indicated.
Embodiment XXXIII
The compound of Embodiment I, wherein Y.sup.2 is CH, CR.sup.a, or
N, and Z is H,
##STR00027## halo, or OH.
Embodiment XXXIV
The compound of Embodiment I, wherein ring A is an unsubstituted or
substituted 5-membered heteroaryl ring.
Embodiment XXXV
The compound of Embodiments I through VII wherein Z is
##STR00028##
In another aspect, the present invention is directed to BET
bromodomain inhibitors having a structural formula (I):
##STR00029## wherein
X is N(R.sup.a1), O, or S;
Y.sup.1 and Y.sup.3, independently, are CH or N;
Y.sup.2 is CR.sup.2, N, or null;
Z is H,
##STR00030## halo, or OH;
A is an unsubstituted or substituted 5-membered heterocyclic
ring;
B is aryl, CH(R.sup.a2)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a2)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a2)-heteroaryl, C.sub.3-10heterocycloalkyl, or
CH(R.sup.a2)--C.sub.3-10heterocycloalkyl, each unsubstituted or
substituted;
G is N, O, or S;
L is null, H, or C(R.sup.d).sub.3;
R.sup.1 is H, halo, OH, OR.sup.a3, R.sup.a3, or
N(R.sup.a3).sub.2;
R.sup.a3, R.sup.a2, R.sup.a3, R.sup.a3, and R.sup.a5 each
independently, is H, C.sub.1-3alkyl, phenyl, or benzyl;
R.sup.2, independently, is H, C.sub.1-3alkyl, phenyl,
(CH.sub.2).sub.1-3C.sub.4-7heterocycloalkyl,
C.sub.4-7heterocycloalkyl, CO.sub.2H, CO.sub.2(C.sub.1-3alkyl),
NH.sub.2, NH(C.sub.1-3alkyl), N(C.sub.1-3alkyl).sub.2,
(CH.sub.2).sub.1-3NMe.sub.2, (CH.sub.2).sub.1-3OH, CH(Me)OH,
C(Me).sub.2NH.sub.2, C(Me).sub.2OH, phenyl, benzyl,
--C(.dbd.O)OR.sup.a4, --C(.dbd.O)N(R.sup.a4).sub.2,
##STR00031##
R.sup.b, independently, is C.sub.1-6alkyl, halo, aryl,
unsubstituted or substituted CH.sub.2-aryl, unsubstituted or
substituted C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, CF.sub.3, CN, OR.sup.a5,
N(R.sup.a5).sub.2, N(CH.sub.3)C(.dbd.O)(C.sub.1-3alkyl),
NH(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
C.sub.3-10heterocycloalkyl,
O(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
O(CH.sub.2).sub.2-3--C.sub.3-10heterocycloalkyl, oxo(.dbd.O), or
CHO;
n is an integer 0, 1, 2, or 3;
R.sup.c and R.sup.d, each independently, are hydrogen,
C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted CH.sub.2-aryl, unsubstituted or substituted
C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, or unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl;
or a pharmaceutically acceptable salt, hydrate, or solvate
thereof.
The compounds of structural formula (I) inhibit BET bromodomains
and are useful in the treatment of a variety of diseases and
conditions. In particular, the compounds of structural formula (I)
are used in methods of treating a disease or condition wherein
inhibition of BET bromodomains provides a benefit, for example,
cancers and proliferative diseases. The method comprises
administering a therapeutically effective amount of a compound of
structural formula (I) to an individual in need thereof. The
present methods also encompass administering a second therapeutic
agent to the individual in addition to the compound of structural
formula (I). The second therapeutic agent is selected from drugs
known as useful in treating the disease or condition afflicting the
individual in need thereof, e.g., a chemotherapeutic agent and/or
radiation known as useful in treating a particular cancer.
As used herein, the term "alkyl" refers to straight chained and
branched saturated C.sub.1-10 hydrocarbon groups, including but not
limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl,
t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl,
2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
3,3-dimethylbutyl, and 2-ethylbutyl. The term C.sub.n means the
alkyl group has "n" carbon atoms. The term "alkylene" refers to an
alkyl group having a substituent. An alkyl, e.g., methyl, or
alkylene, e.g., --CH.sub.2--, group can be substituted with one or
more, and typically one to three, of independently selected halo,
trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano,
alkylamino, or amino groups, for example.
As used herein, the term "halo" is defined as fluoro, chloro,
bromo, and iodo.
The term "hydroxy" is defined as --OH.
The term "alkoxy" is defined as --OR, wherein R is alkyl.
The term "amino" is defined as --NH.sub.2, and the term
"alkylamino" is defined as --NR.sub.2, wherein at least one R is
alkyl and the second R is alkyl or hydrogen.
The term "carbamoyl" is defined as --C(.dbd.O)NR.sub.2.
The term "carboxy" is defined as --C(.dbd.O)OH or a salt
thereof.
The term "nitro" is defined as --NO.sub.2.
The term "cyano" is defined as --CN.
The term "trifluoromethyl" is defined as --CF.sub.3.
The term "trifluoromethoxy" is defined as --OCF.sub.3.
The term "Ts" means tosylate
##STR00032##
The term "Bn" means benzyl
##STR00033##
As used herein, groups such as
##STR00034## is an abbreviation for
##STR00035##
As used herein, the term "aryl" refers to a monocyclic or
polycyclic aromatic group, preferably a monocyclic or bicyclic
aromatic group. Examples of aryl groups include, but are not
limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl,
phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to
bicyclic and tricyclic carbon rings, where one ring is aromatic and
the others are saturated, partially unsaturated, or aromatic, for
example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl
(tetralinyl). Unless otherwise indicated, an aryl group can be
unsubstituted or substituted with one or more, and in particular
one to four, groups independently selected from, for example, halo,
alkyl, alkenyl, --OCF.sub.3, --NO.sub.2, --CN, --NC, --OH, alkoxy,
amino, alkylamino, --CO.sub.2H, --CO.sub.2alkyl, --OCOalkyl, aryl,
and heteroaryl.
As used herein, the term "heterocyclic" refers to a heteroaryl and
heterocycloalkyl ring systems.
As used herein, the term "heteroaryl" refers to a monocyclic or
bicyclic ring system containing one or two aromatic rings and
containing at least one nitrogen, oxygen, or sulfur atom in an
aromatic ring. Each ring of a heteroaryl group can contain one or
two O atoms, one or two S atoms, and/or one to four N atoms,
provided that the total number of heteroatoms in each ring is four
or less and each ring contains at least one carbon atom. In certain
embodiments, the heteroaryl group has from 5 to 20, from 5 to 15,
or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl
groups include, but are not limited to, furanyl, imidazolyl,
isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl,
pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and
triazolyl. Examples of bicyclic heteroaryl groups include, but are
not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl,
benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,
benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl,
imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl,
indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl,
isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl,
phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl,
quinolinyl, quinoxalinyl, quiazolinyl, thiadiazolopyrimidyl, and
thienopyridyl. Unless otherwise indicated, a heteroaryl group can
be unsubstituted or substituted with one or more, and in particular
one to four, substituents selected from, for example, halo, alkyl,
alkenyl, --OCF.sub.3, --NO.sub.2, --CN, NC, --OH, alkoxy, amino,
alkylamino, --CO.sub.2H, --CO.sub.2alkyl, --OCOalkyl, aryl, and
heteroaryl.
As used herein, the term "cycloalkyl" means a monocyclic aliphatic
ring containing three to eight carbon atoms, including cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl,
optionally substituted with one or more, and typically one to
three, of independently selected halo, trifluoromethyl,
trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or
amino groups, for example.
As used herein, the term "heterocycloalkyl" means a monocyclic or a
bicyclic aliphatic ring containing 4 to 12 total atoms, of which
one to five of the atoms are independently selected from nitrogen,
oxygen, and sulfur and the remaining atoms are carbon. Nonlimiting
examples of heterocycloalkyl groups are azetidinyl, pyrrolidinyl,
piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl,
thiomorpholinyl, dihydropyridinyl, oxacycloheptyl,
dioxacycloheptyl, thiacycloheptyl, diazacycloheptyl, each
optionally substituted with one or more, and typically one to
three, of independently selected halo, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C.sub.2-7 alkenyl,
C.sub.2-7 alkynyl, or the like on an atom of the ring.
The term "alkenyl" is defined identically as "alkyl," except for
containing a carbon-carbon double bond, e.g., ethenyl, propenyl,
and butenyl. The term "alkynyl" is defined identically as "alkyl,"
except the group contains a carbon-carbon triple bond.
As used herein, the term "C.sub.1-6hydroxyalkyl" refers to straight
chained and branched saturated C.sub.1-6 hydrocarbon groups
substituted with one, two, three, or four hydroxy groups. In one
embodiment, the C.sub.1-6hydroxyalkyl is substituted with one
hydroxy group. In one embodiment, the C.sub.1-6hydroxyalkyl is
substituted with two hydroxy groups. Examples of C.sub.1-6
hydroxyalkyl groups include, but are not limited to,
--C(CH.sub.3).sub.2OH, --C(H)(CH.sub.3).sub.3CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, and
--CH.sub.2CH.sub.2CH(OH)CH.sub.2OH.
As used herein, the term "hydroxyC.sub.3-10heterocycloalkyl" refers
to a heterocycloalkyl group as defined above substituted with one
or two hydroxy groups. In one embodiment, the
hydroxyC.sub.3-10heterocycloalkyl is substituted with one hydroxy
group. Examples of hydroxyC.sub.3-10heterocycloalkyl groups
include, but are not limited to, piperidin-4-ol and
pyrrolidin-3-ol.
As used herein the term "hydroxycycloalkyl" refers to a cycloalkyl
group as defined above substituted with one or two hydroxy groups.
In one embodiment, the hydroxycycloalkyl is substituted with one
hydroxy group.
The term "pharmaceutically acceptable anion" as used herein refers
to an anion associated with a quaternary pyridinium of the present
disclosure that is acceptable for administration to a patient,
e.g., a mammal, e.g., a human. In one embodiment, the
pharmaceutically acceptable anion is the anion of a
pharmaceutically acceptable inorganic acid, e.g., hydrochloric,
perchloric, sulfuric, phosphoric, hydrobromic, hydroiodic or nitric
acid and the like. In one embodiment, the pharmaceutically
acceptable anion is the anion of a pharmaceutically acceptable
organic acid, e.g., a mono or polyvalent organic acid, e.g.,
citric, fumaric, maleic, malic, ascorbic, succinic, tartaric,
benzoic, acetic, phenylacetic, methanesulfonic, ethansulfonic,
benzenesulfonic or p-toluenesulfonic acid and the like.
In accordance with the present invention, ring A is a five-membered
heterocyclic ring, either heteroaryl or heterocycloalkyl,
containing one to four heteroatoms, i.e., independently are
nitrogen, oxygen, or sulfur. In various embodiments, ring A is
substituted with one to three groups, independently selected from
the group consisting of alkyl, cycloalkyl, haloalkyl, and
halocycloalkyl, for example, methyl, ethyl, isopropyl,
trifluoromethyl, cyclopropyl, or cyclobutyl.
Nonlimiting examples of A rings include, but are not limited to
##STR00036## wherein R.sup.a is H, C.sub.1-3alkyl, cyclopropyl,
cyclobutyl, phenyl, or benzyl, and each optionally substituted with
one to three substituents.
In some preferred embodiments, the A ring is:
##STR00037## optionally substituted with one or more methyl and/or
ethyl groups.
In some specific embodiments, the A ring is:
##STR00038##
In some embodiments X is --NH, --NC.sub.6H.sub.5, --NCH.sub.3, or
--NCH.sub.2C.sub.6H.sub.5.
In some embodiments Y.sup.1 and Y.sup.3 are N and N, CH and CH, or
CH and N.
In some embodiments, Y.sup.2 is CH, N, CCH.sub.3,
CCH(CH.sub.3).sub.2,
##STR00039## C--CO.sub.2H, C--CO.sub.2CH.sub.3,
##STR00040## CCH.sub.2OH, CCH.sub.2NH.sub.2,
CCH.sub.2N(CH.sub.3).sub.2,
##STR00041## C(CH.sub.2).sub.2N(CH.sub.3).sub.2,
C(CH.sub.2).sub.2OH,
##STR00042##
In some preferred embodiments R.sup.1 is H or --OCH.sub.3.
In various embodiments, the ring system
##STR00043## is
##STR00044## ##STR00045## wherein R.sup.a4, independently, is H,
C.sub.1-3alkyl, phenyl, or benzyl.
In various embodiments, Z is
##STR00046##
Nonlimiting examples of the B ring include
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## wherein R.sup.a5 is H, C.sub.1-3alkyl, phenyl, or
benzyl.
Various substituents on the carbon atoms of the B ring include, but
are not limited to, one to three of methyl, phenyl, benzyl, CHO,
CF.sub.3, OCH.sub.3, fluoro, pyridinyl, chloro, isopropyl,
cyclopropyl, ethyl, C(CH.sub.3)OH, NH.sub.2, N(CH.sub.3).sub.2,
N(C.sub.2H.sub.5).sub.2, NH(CH.sub.2).sub.2N(CH.sub.3).sub.2,
N(CH.sub.3)C(.dbd.O)CH.sub.3, oxo(.dbd.O), OH, OCH(CH.sub.3).sub.2,
O(CH.sub.2).sub.2N(CH.sub.3).sub.2, pyrrolyl, piperidinyl,
piperizinyl, morpholino,
##STR00053##
In other embodiments, Z is N(CH.sub.3).sub.2, H, OH, or chloro.
Additionally, salts, hydrates, and solvates of the present
compounds also are included in the present invention and can be
used in the methods disclosed herein. The present invention further
includes all possible stereoisomers and geometric isomers of the
compounds of structural formula (I). The present invention includes
both racemic compounds and optically active isomers. When a
compound of structural formula (I) is desired as a single
enantiomer, it can be obtained either by resolution of the final
product or by stereospecific synthesis from either isomerically
pure starting material or use of a chiral auxiliary reagent, for
example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages
883-888 (1997). Resolution of the final product, an intermediate,
or a starting material can be achieved by any suitable method known
in the art. Additionally, in situations where tautomers of the
compounds of structural formula (I) are possible, the present
invention is intended to include all tautomeric forms of the
compounds.
Compounds of the invention can exist as salts. Pharmaceutically
acceptable salts of the compounds of the invention often are
preferred in the methods of the invention. As used herein, the term
"pharmaceutically acceptable salts" refers to salts or zwitterionic
forms of the compounds of structural formula (I). Salts of
compounds of formula (I) can be prepared during the final isolation
and purification of the compounds or separately by reacting the
compound with an acid having a suitable cation. The
pharmaceutically acceptable salts of compounds of structural
formula (I) can be acid addition salts formed with pharmaceutically
acceptable acids. Examples of acids which can be employed to form
pharmaceutically acceptable salts include inorganic acids such as
nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric,
and organic acids such as oxalic, maleic, succinic, and citric.
Nonlimiting examples of salts of compounds of the invention
include, but are not limited to, the hydrochloride, hydrobromide,
hydroiodide, sulfate, bisulfate, 2-hydroxyethanesulfonate,
phosphate, hydrogen phosphate, acetate, adipate, alginate,
aspartate, benzoate, bisulfate, butyrate, camphorate,
camphorsulfonate, digluconate, glycerolphsphate, hemisulfate,
heptanoate, hexanoate, formate, succinate, fumarate, maleate,
ascorbate, isethionate, salicylate, methanesulfonate,
mesitylenesulfonate, naphthylenesulfonate, nicotinate,
2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,
3-phenylproprionate, picrate, pivalate, propionate,
trichloroacetate, trifluoroacetate, phosphate, glutamate,
bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate,
tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene
sulphonate, and p-toluenesulfonate salts. In addition, available
amino groups present in the compounds of the invention can be
quaternized with methyl, ethyl, propyl, and butyl chlorides,
bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl
sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides,
and iodides; and benzyl and phenethyl bromides. In light of the
foregoing, any reference to compounds of the present invention
appearing herein is intended to include compounds of structural
formula (I) as well as pharmaceutically acceptable salts, hydrates,
or solvates thereof.
Specific compounds of the present invention include, but are not
limited to, compounds having the structure set forth below.
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064##
Additional compounds of the present invention include, but are not
limited to, compounds having the structure set forth below:
##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069##
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092##
Additional compounds of the present invention include, but are not
limited to, compounds having the structure set forth below:
##STR00093##
In one embodiment, the present invention provides compounds having
Formula (II):
##STR00094## wherein R.sup.2 is H, C.sub.1-3alkyl, phenyl,
(CH.sub.2).sub.1-3C.sub.4-7heterocycloalkyl,
C.sub.4-7heterocycloalkyl, CO.sub.2H, CO.sub.2(C.sub.1-3alkyl),
NH.sub.2, NH(C.sub.1-3alkyl), N(C.sub.1-3alkyl).sub.2,
(CH.sub.2).sub.1-3NMe.sub.2, (CH.sub.2).sub.1-3OH, CH(Me)OH,
C(Me).sub.2NH.sub.2, C(Me).sub.2OH, phenyl, benzyl,
--C(.dbd.O)OR.sup.a4, --C(.dbd.O)N(R.sup.a4).sub.2,
##STR00095## B is aryl, CH(R.sup.a2)-aryl, C.sub.3-10cycloalkyl,
CH(R.sup.a2)--C.sub.3-10cycloalkyl, heteroaryl,
CH(R.sup.a2)-heteroaryl, C.sub.3-10heterocycloalkyl, or
CH(R.sup.a2)--C.sub.3-10heterocycloalkyl, each unsubstituted or
substituted; and R.sup.b is C.sub.1-6alkyl, halo, aryl,
unsubstituted or substituted CH.sub.2-aryl, unsubstituted or
substituted C.sub.3-10cycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10cycloalkyl, heteroaryl, unsubstituted or
substituted CH.sub.2-heteroaryl, unsubstituted or substituted
C.sub.3-10heterocycloalkyl, unsubstituted or substituted
CH.sub.2--C.sub.3-10heterocycloalkyl, CF.sub.3, CN, OR.sup.a3,
N(R.sup.a5).sub.2, N(CH.sub.3)C(.dbd.O)(C.sub.1-3alkyl),
NH(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
C.sub.3-10heterocycloalkyl,
O(CH.sub.2).sub.2-3N(C.sub.1-3alkyl).sub.2,
O(CH.sub.2).sub.2-3--C.sub.3-10heterocycloalkyl, oxo(.dbd.O), or
CHO; and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt,
hydrate, or solvate thereof.
In another embodiment, the present invention provides compounds
having Formula (II), wherein B is heteroaryl and n is 0, or a
pharmaceutically acceptable salt, hydrate, or solvate thereof. In
another embodiment, B is:
##STR00096##
In another embodiment, the present invention provides compounds
having Formula (II), wherein B is heteroaryl and n is 1, or a
pharmaceutically acceptable salt, hydrate, or solvate thereof. In
another embodiment, B is:
##STR00097##
In another embodiment, the present invention provides a compound
selected from the group consisting of:
##STR00098## ##STR00099## or a pharmaceutically acceptable salt,
hydrate, or solvate thereof.
The present invention provides BET bromodomain inhibitors, as
exemplified by compounds of structural formula (I), for the
treatment of a variety of diseases and conditions wherein
inhibition of BET brodomains has a beneficial effect. In one
embodiment, the present invention relates to a method of treating
an individual suffering from a disease or condition wherein
inhibition of the BET bromodomains provides a benefit comprising
administering a therapeutically effective amount of a compound of
structural formula (I) to an individual in need thereof.
The method of the present invention can be accomplished by
administering a compound of structural formula (I) as the neat
compound or as a pharmaceutical composition. Administration of a
pharmaceutical composition, or neat compound of structural formula
(I), can be performed during or after the onset of the disease or
condition of interest. Typically, the pharmaceutical compositions
are sterile, and contain no toxic, carcinogenic, or mutagenic
compounds that would cause an adverse reaction when administered.
Further provided are kits comprising a compound of structural
formula (I) and, optionally, a second therapeutic agent useful in
the treatment of diseases and conditions wherein inhibition of BET
bromodomains provides a benefit, packaged separately or together,
and an insert having instructions for using these active
agents.
In many embodiments, a compound of structural formula (I) is
administered in conjunction with a second therapeutic agent useful
in the treatment of a disease or condition wherein inhibition of
BET bromodomains provides a benefit. The second therapeutic agent
is different from the compound of structural formula (I). A
compound of structural formula (I) and the second therapeutic agent
can be administered simultaneously or sequentially to achieve the
desired effect. In addition, the compound of structural formula (I)
and second therapeutic agent can be administered from a single
composition or two separate compositions.
The second therapeutic agent is administered in an amount to
provide its desired therapeutic effect. The effective dosage range
for each second therapeutic agent is known in the art, and the
second therapeutic agent is administered to an individual in need
thereof within such established ranges.
A compound of structural formula (I) and the second therapeutic
agent can be administered together as a single-unit dose or
separately as multi-unit doses, wherein the compound of structural
formula (I) is administered before the second therapeutic agent or
vice versa. One or more dose of the compound of structural formula
(I) and/or one or more dose of the second therapeutic agent can be
administered. The compounds of structural formula (I) therefore can
be used in conjunction with one or more second therapeutic agents,
for example, but not limited to, anticancer agents.
Diseases and conditions treatable by a method of the present
invention include, but are not limited to, cancer and other
proliferative disorders, inflammatory diseases, sepsis, autoimmune
disease, and viral infection. In one embodiment, a human patient is
treated with a compound of structural formula (I) and an optional
pharmaceutically acceptable carrier, adjuvant, or vehicle, wherein
the compound is administered in an amount sufficient to inhibit BET
bromodomain activity in the patient.
In one embodiment, the disease to be treated by a compound and
method of the present invention is cancer. Examples of treatable
cancers include, but are not limited to, adrenal cancer, acinic
cell carcinoma, acoustic neuroma, acral lentigious melanoma,
acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia,
acute lymphoblastic leukemia, acute megakaryoblastic leukemia,
acute monocytic leukemia, actue promyelocytic leukemia,
adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid
odontogenic tumor, adenosquamous carcinoma, adipose tissue
neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma,
aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar
rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma,
anaplastic large cell lymphoma, anaplastic thyroid cancer,
angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma,
astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic
lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell
lymphoma, basal cell carcinoma, biliary tract cancer, bladder
cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,
Burkitt's lymphoma, breast cancer, brain cancer, carcinoma,
carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma,
myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid
plexus papilloma, clear-cell sarcoma of the kidney,
craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer,
colorectal cancer, Degos disease, desmoplastic small round cell
tumor, diffuse large B-cell lymphoma, dysembryoplastic
neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine
gland neoplasm, endodermal sinus tumor, enteropathy-associated
T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma,
fibrosarcoma, follicular lymphoma, follicular thyroid cancer,
ganglioneuroma, gastrointestinal cancer, germ cell tumor,
gestational choriocarcinoma, giant cell fibroblastoma, giant cell
tumor of the bone, glial tumor, glioblastoma multiforme, glioma,
gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell
tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy
cell leukemia, hemangioblastoma, head and neck cancer,
hemangiopericytoma, hematological malignancy, hepatoblastoma,
hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, invasive lobular carcinoma, intestinal cancer, kidney
cancer, laryngeal cancer, lentigo maligna, lethal midline
carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer,
lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute
lymphocytic leukemia, acute myelogeous leukemia, chronic
lymphocytic leukemia, liver cancer, small cell lung cancer,
non-small cell lung cancer, MALT lymphoma, malignant fibrous
histiocytoma, malignant peripheral nerve sheath tumor, malignant
triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma,
mast cell leukemia, mediastinal germ cell tumor, medullary
carcinoma of the breast, medullary thyroid cancer, medulloblastoma,
melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic
urothelial carcinoma, mixed Mullerian tumor, mucinous tumor,
multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid
liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma,
neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma,
ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma,
optic nerve sheath meningioma, optic nerve tumor, oral cancer,
osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid
cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma,
pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma,
precursor T-lymphoblastic lymphoma, primary central nervous system
lymphoma, primary effusion lymphoma, preimary peritoneal cancer,
prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma
periotonei, renal cell carcinoma, renal medullary carcinoma,
retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's
transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma,
Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring
cell carcinoma, skin cancer, small blue round cell tumors, small
cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart,
spinal tumor, splenic marginal zone lymphoma, squamous cell
carcinoma, synovial sarcoma, Sezary's disease, small intestine
cancer, squamous carcinoma, stomach cancer, T-cell lymphoma,
testicular cancer, thecoma, thyroid cancer, transitional cell
carcinoma, throat cancer, urachal cancer, urogenital cancer,
urothelial carcinoma, uveal melanoma, uterine cancer, verrucous
carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer,
Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'
tumor.
In another embodiment, the present invention provides a method of
treating a benign proliferative disorder, such as, but are not
limited to, benign soft tissue tumors, bone tumors, brain and
spinal tumors, eyelid and orbital tumors, granuloma, lipoma,
meningioma, multiple endocrine neoplasia, nasal polyps, pituitary
tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses,
stomach polyps, thyroid nodules, cystic neoplasms of the pancreas,
hemangiomas, vocal cord nodules, polyps, and cysts, Castleman
disease, chronic pilonidal disease, dermatofibroma, pilar cyst,
pyogenic granuloma, and juvenile polyposis syndrome.
The compounds and methods of the present invention also treat
infectious and noninfectious inflammatory events and autoimmune and
other inflammatory diseases by administration of an effective
amount of a present compound to a mammal, in particular a human in
need of such treatment. Examples of autoimmune and inflammatory
diseases, disorders, and syndromes treated using the compounds and
methods described herein include inflammatory pelvic disease,
urethritis, skin sunburn, sinusitis, pneumonitis, encephalitis,
meningitis, myocarditis, nephritis, osteomyelitis, myositis,
hepatitis, gastritis, enteritis, dermatitis, gingivitis,
appendictitis, pancreatitis, cholocystitus, agammaglobulinemia,
psoriasis, allergy, Crohn's disease, irrtiable bowel syndrome,
ulcerative colitis, Sjogren's disease, tissue graft rejection,
hyperacute rejection of transplanted organs, asthma, allergic
rhinitis, chronic obstructive pulmonary disease (COPD), autoimmune
polyglandular disease (also known as autoimmune polyglandular
syndrome), autoimmune alopecia, pernicious anemia,
glomerulonephritis, dermatomyositis, multiple sclerosis,
scleroderma, vasculitis, autoimmune hemolytic and thrombocytopenic
states, Goodpasture's syndrome, athersclerosis, Addison's disease,
Parkinson's disease, Alzheimer's disease, Type I diabetes, septic
shock, systemic lupus erythematosus (SLE), rheumatoid arthritis,
psoriatic arthritis, juvenile arthritis, osteoarthritis, chronic
idiopathic thrombocytopenic purpura, Waldenstrom macroglobulinemia,
myasthenia gravis, Hashimoto's thyroiditis, atopic dermatitis,
degenerative joint disease, vitiligo, autoimmune hypopituatarism,
Guillain-Barre syndrome, Behcet's disease, scleracierma, mycosis
fungoides, acute inflammatory responses (such as acute respiratory
distress syndrome and ischemia/reperfusion injury), and Graves'
disease.
In other embodiments, the present invention provides a method of
treating systemic inflammatory response syndromes, such as
LPS-induced endotoxic shock and/or bacteria-induced sepsis by
administration of an effective amount of a present compound to a
mammal, in particular a human in need of such treatment.
The invention further provides a method for treating viral
infections and diseases. Examples of viral infections and diseases
treated using the compounds and methods described herein include
episome-based DNA viruses including, but not limited to, human
papillomavirus, Herpesvirus, Epstein-Barr virus, human
immunodeficiency virus, hepatitis B virus, and hepatitis C
virus.
In another embodiment, a therapeutic method of modulating protein
methylation, gene expression, cell proliferation, cell
differentiation and/or apoptosis in vivo in diseases mentioned
above, in particular cancer, inflammatory disease, and/or viral
disease is provided by administering a therapeutically effective
amount of one or more BET inhibitor of structural formula (I) to a
subject in need of such therapy.
The invention further provides a method of regulating endogenous or
heterologous promoter activity by contacting a cell with a provided
compound.
In the present methods, a therapeutically effective amount of one
or more compound (I), typically formulated in accordance with
pharmaceutical practice, is administered to a human being in need
thereof. Whether such a treatment is indicated depends on the
individual case and is subject to medical assessment (diagnosis)
that takes into consideration signs, symptoms, and/or malfunctions
that are present, the risks of developing particular signs,
symptoms and/or malfunctions, and other factors.
A compound of structural formula (I) can be administered by any
suitable route, for example by oral, buccal, inhalation,
sublingual, rectal, vaginal, intracisternal or intrathecal through
lumbar puncture, transurethral, nasal, percutaneous, i.e.,
transdermal, or parenteral (including intravenous, intramuscular,
subcutaneous, intracoronary, intradermal, intramammary,
intraperitoneal, intraarticular, intrathecal, retrobulbar,
intrapulmonary injection and/or surgical implantation at a
particular site) administration. Parenteral administration can be
accomplished using a needle and syringe or using a high pressure
technique.
Pharmaceutical compositions include those wherein a compound of
structural formula (I) is administered in an effective amount to
achieve its intended purpose. The exact formulation, route of
administration, and dosage is determined by an individual physician
in view of the diagnosed condition or disease. Dosage amount and
interval can be adjusted individually to provide levels of a
compound of structural formula (I) that is sufficient to maintain
therapeutic effects.
Toxicity and therapeutic efficacy of the compounds of structural
formula (I) can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
maximum tolerated dose (MTD) of a compound, which defines as the
highest dose that causes no toxicity in animals. The dose ratio
between the maximum tolerated dose and therapeutic effects (e.g.
inhibiting of tumor growth) is the therapeutic index. The dosage
can vary within this range depending upon the dosage form employed,
and the route of administration utilized. Determination of a
therapeutically effective amount is well within the capability of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
A therapeutically effective amount of a compound of structural
formula (I) required for use in therapy varies with the nature of
the condition being treated, the length of time that activity is
desired, and the age and the condition of the patient, and
ultimately is determined by the attendant physician. Dosage amounts
and intervals can be adjusted individually to provide plasma levels
of the BET bromodomain inhibitor that are sufficient to maintain
the desired therapeutic effects. The desired dose conveniently can
be administered in a single dose, or as multiple doses administered
at appropriate intervals, for example as one, two, three, four or
more subdoses per day. Multiple doses often are desired, or
required. For example, a present BET bromodomain inhibitor can be
administered at a frequency of: four doses delivered as one dose
per day at four-day intervals (q4d.times.4); four doses delivered
as one dose per day at three-day intervals (q3d.times.4); one dose
delivered per day at five-day intervals (qd.times.5); one dose per
week for three weeks (qwk3); five daily doses, with two days rest,
and another five daily doses (5/2/5); or, any dose regimen
determined to be appropriate for the circumstance.
A compound of structural formula (I) used in a method of the
present invention can be administered in an amount of about 0.005
to about 500 milligrams per dose, about 0.05 to about 250
milligrams per dose, or about 0.5 to about 100 milligrams per dose.
For example, a compound of structural formula (I) can be
administered, per dose, in an amount of about 0.005, 0.05, 0.5, 5,
10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500
milligrams, including all doses between 0.005 and 500
milligrams.
The dosage of a composition containing a BET bromodomain inhibitor
of structural formula (I), or a composition containing the same,
can be from about 1 ng/kg to about 200 mg/kg, about 1 .mu.g/kg to
about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg. The dosage of
a composition can be at any dosage including, but not limited to,
about 1 .mu.g/kg. The dosage of a composition may be at any dosage
including, but not limited to, about 1 .mu.g/kg, 10 .mu.g/kg, 25
.mu.g/kg, 50 .mu.g/kg, 75 .mu.g/kg, 100 .mu.g/kg, 125 .mu.g/kg, 150
.mu.g/kg, 175 .mu.g/kg, 200 .mu.g/kg, 225 .mu.g/kg, 250 .mu.g/kg,
275 .mu.g/kg, 300 .mu.g/kg, 325 .mu.g/kg, 350 .mu.g/kg, 375
.mu.g/kg, 400 .mu.g/kg, 425 .mu.g/kg, 450 .mu.g/kg, 475 .mu.g/kg,
500 .mu.g/kg, 525 .mu.g/kg, 550 .mu.g/kg, 575 .mu.g/kg, 600
.mu.g/kg, 625 .mu.g/kg, 650 .mu.g/kg, 675 .mu.g/kg, 700 .mu.g/kg,
725 .mu.g/kg, 750 .mu.g/kg, 775 .mu.g/kg, 800 .mu.g/kg, 825
.mu.g/kg, 850 .mu.g/kg, 875 .mu.g/kg, 900 .mu.g/kg, 925 .mu.g/kg,
950 .mu.g/kg, 975 .mu.g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg,
20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50
mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125
mg/kg, 150 mg/kg, 175 mg/kg, or 200 mg/kg. The above dosages are
exemplary of the average case, but there can be individual
instances in which higher or lower dosages are merited, and such
are within the scope of this invention. In practice, the physician
determines the actual dosing regimen that is most suitable for an
individual patient, which can vary with the age, weight, and
response of the particular patient.
As stated above, a BET brodomomain inhibitor of structural formula
(I) can be administered in combination with a second
therapeutically active agent. In some embodiments, the second
therapeutic agent is an epigenetic drug. As used herein, the term
"epigenetic drug" refers to a therapeutic agent that targets an
epigenetic regulator. Examples of epigenetic regulators include the
histone lysine methyltransferases, histone arginine methyl
transferases, histone demethylases, histone deacetylases, histone
acetylases, and DNA methyltransferases. Histone deacetylase
inhibitors include, but are not limited to, vorinostat.
In another embodiment, chemotherapeutic agents or other
anti-proliferative agents can be combined with a present BET
bromodomain inhibitor to treat proliferative diseases and cancer.
Examples of therapies and anticancer agents that can be used in
combination with compounds of structural formula (I) include
surgery, radiotherapy (e.g., gamma-radiation, neutron beam
radiotherapy, electron beam radiotherapy, proton therapy,
brachytherapy, and systemic radioactive isotopes), endocrine
therapy, a biologic response modifier (e.g., an interferon, an
interleukin, tumor necrosis factor (TNF), hyperthermia and
cryotherapy, an agent to attenuate any adverse effect (e.g., an
antiemetic), and any other approved chemotherapeutic drug.
Examples of antiproliferative compounds include, but are not
limited to, an aromatase inhibitor; an anti-estrogen; an
anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor;
a topoisomerase II inhibitor; a microtubule active agent; an
alkylating agent; a retinoid, a carontenoid, or a tocopherol; a
cyclooxygenase inhibitor; an MMP inhibitor; an mTOR inhibitor; an
antimetabolite; a platin compound; a methionine aminopeptidase
inhibitor; a bisphosphonate; an antiproliferative antibody; a
heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a
telomerase inhibitor; a proteasome inhibitor; a compound used in
the treatment of hematologic malignancies; a Flt-3 inhibitor; an
Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK
inhibitor; an antitumor antibiotic; a nitrosourea; a compound
targeting/decreasing protein or lipid kinase activity, a compound
targeting/decreasing protein or lipid phosphatase activity, or any
further anti-angiogenic compound.
Nonlimiting exemplary aromatase inhibitors include, but are not
limited to, steroids, such as atamestane, exemestane, and
formestane, and non-steroids, such as aminoglutethimide,
roglethimide, pyridoglutethimide, trilostane, testolactone,
ketokonazole, vorozole, fadrozole, anastrozole, and letrozole.
Nonlimiting anti-estrogens include, but are not limited to,
tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride.
Anti-androgens include, but are not limited to, bicalutamide.
Gonadorelin agonists include, but are not limited to, abarelix,
goserelin, and goserelin acetate.
Exemplary topoisomerase I inhibitors include, but are not limited
to, topotecan, gimatecan, irinotecan, camptothecin and its
analogues, 9-nitrocamptothecin, and the macromolecular camptothecin
conjugate PNU-166148. Topoisomerase II inhibitors include, but are
not limited to, anthracyclines, such as doxorubicin, daunorubicin,
epirubicin, idarubicin, and nemorubicin; anthraquinones, such as
mitoxantrone and losoxantrone; and podophillotoxines, such as
etoposide and teniposide.
Microtubule active agents include microtubule stabilizing,
microtubule destabilizing compounds, and microtublin polymerization
inhibitors including, but not limited to, taxanes, such as
paclitaxel and docetaxel; vinca alkaloids, such as vinblastine,
vinblastine sulfate, vincristine, and vincristine sulfate, and
vinorelbine; discodermolides; cochicine and epothilones and
derivatives thereof.
Exemplary nonlimiting alkylating agents include cyclophosphamide,
ifosfamide, melphalan, and nitrosoureas, such as carmustine and
lomustine.
Exemplary nonlimiting cyclooxygenase inhibitors include Cox-2
inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and
derivatives, such as celecoxib, rofecoxib, etoricoxib, valdecoxib,
or a 5-alkyl-2-arylaminophenylacetic acid, such as lumiracoxib.
Exemplary nonlimiting matrix metalloproteinase inhibitors ("MMP
inhibitors") include collagen peptidomimetic and nonpeptidomimetic
inhibitors, tetracycline derivatives, batimastat, marimastat,
prinomastat, metastat, BMS-279251, BAY 12-9566, TAA211, MMI270B,
and AAJ996.
Exemplary nonlimiting mTOR inhibitors include compounds that
inhibit the mammalian target of rapamycin (mTOR) and possess
antiproliferative activity such as sirolimus, everolimus, CCI-779,
and ABT578.
Exemplary nonlimiting antimetabolites include 5-fluorouracil
(5-FU), capecitabine, gemcitabine, DNA demethylating compounds,
such as 5-azacytidine and decitabine, methotrexate and edatrexate,
and folic acid antagonists, such as pemetrexed.
Exemplary nonlimiting platin compounds include carboplatin,
cis-platin, cisplatinum, and oxaliplatin.
Exemplary nonlimiting methionine aminopeptidase inhibitors include
bengamide or a derivative thereof and PPI-2458.
Exemplary nonlimiting bisphosphonates include etridonic acid,
clodronic acid, tiludronic acid, pamidronic acid, alendronic acid,
ibandronic acid, risedronic acid, and zoledronic acid.
Exemplary nonlimiting antiproliferative antibodies include
trastuzumab, trastuzumab-DM1, cetuximab, bevacizumab, rituximab,
PR064553, and 2C4. The term"antibody" is meant to include intact
monoclonal antibodies, polyclonal antibodies, multispecific
antibodies formed from at least two intact antibodies, and antibody
fragments, so long as they exhibit the desired biological
activity.
Exemplary nonlimiting heparanase inhibitors include compounds that
target, decrease, or inhibit heparin sulfate degradation, such as
P1-88 and OGT2115.
The term "an inhibitor of Ras oncogenic isoforms," such as H-Ras,
K-Ras, or N-Ras, as used herein refers to a compound which targets,
decreases, or inhibits the oncogenic activity of Ras, for example,
a farnesyl transferase inhibitor, such as L-744832, DK8G557,
tipifarnib, and lonafarnib.
Exemplary nonlimiting telomerase inhibitors include compounds that
target, decrease, or inhibit the activity of telomerase, such as
compounds that inhibit the telomerase receptor, such as
telomestatin.
Exemplary nonlimiting proteasome inhibitors include compounds that
target, decrease, or inhibit the activity of the proteasome
including, but not limited to, bortezomid.
The phrase "compounds used in the treatment of hematologic
malignancies" as used herein includes FMS-like tyrosine kinase
inhibitors, which are compounds targeting, decreasing or inhibiting
the activity of FMS-like tyrosine kinase receptors (Flt-3R);
interferon, I-.beta.-D-arabinofuransylcytosine (ara-c), and
bisulfan; and ALK inhibitors, which are compounds which target,
decrease, or inhibit anaplastic lymphoma kinase.
Exemplary nonlimiting Flt-3 inhibitors include PKC412, midostaurin,
a staurosporine derivative, SU11248, and MLN518.
Exemplary nonlimiting HSP90 inhibitors include compounds targeting,
decreasing, or inhibiting the intrinsic ATPase activity of HSP90;
or degrading, targeting, decreasing or inhibiting the HSP90 client
proteins via the ubiquitin proteosome pathway. Compounds targeting,
decreasing or inhibiting the intrinsic ATPase activity of HSP90 are
especially compounds, proteins, or antibodies that inhibit the
ATPase activity of HSP90, such as 17-allylamino,
17-demethoxygeldanamycin (17AAG), a geldanamycin derivative; other
geldanamycin related compounds; radicicol and HDAC inhibitors.
The phrase "a compound targeting/decreasing a protein or lipid
kinase activity; or a protein or lipid phosphatase activity; or any
further anti-angiogenic compound" as used herein includes a protein
tyrosine kinase and/or serine and/or threonine kinase inhibitor or
lipid kinase inhibitor, such as a) a compound targeting,
decreasing, or inhibiting the activity of the platelet-derived
growth factor-receptors (PDGFR), such as a compound that targets,
decreases, or inhibits the activity of PDGFR, such as an
N-phenyl-2-pyrimidine-amine derivatives, such as imatinib, SUlOl,
SU6668, and GFB-111; b) a compound targeting, decreasing, or
inhibiting the activity of the fibroblast growth factor-receptors
(FGFR); c) a compound targeting, decreasing, or inhibiting the
activity of the insulin-like growth factor receptor I (IGF-IR),
such as a compound that targets, decreases, or inhibits the
activity of IGF-IR; d) a compound targeting, decreasing, or
inhibiting the activity of the Trk receptor tyrosine kinase family,
or ephrin B4 inhibitors; e) a compound targeting, decreasing, or
inhibiting the activity of the Axl receptor tyrosine kinase family;
f) a compound targeting, decreasing, or inhibiting the activity of
the Ret receptor tyrosine kinase; g) a compound targeting,
decreasing, or inhibiting the activity of the Kit/SCFR receptor
tyrosine kinase, such as imatinib; h) a compound targeting,
decreasing, or inhibiting the activity of the c-Kit receptor
tyrosine kinases, such as imatinib; i) a compound targeting,
decreasing, or inhibiting the activity of members of the c-Abl
family, their gene-fusion products (e.g. Bcr-Abl kinase) and
mutants, such as an N-phenyl-2-pyrimidine-amine derivative, such as
imatinib or nilotinib; PD180970; AG957; NSC 680410; PD173955; or
dasatinib; j) a compound targeting, decreasing, or inhibiting the
activity of members of the protein kinase C(PKC) and Raf family of
serine/threonine kinases, members of the MEK, SRC, JAK, FAK, PDK1,
PKB/Akt, and Ras/MAPK family members, and/or members of the
cyclin-dependent kinase family (CDK), such as a staurosporine
derivative disclosed in U.S. Pat. No. 5,093,330, incorporated
herein by reference, such as midostaurin; examples of further
compounds include UCN-01, safingol, BAY 43-9006, bryostatin 1,
perifosine; ilmofosine; RO 318220 and RO 320432; GO 6976; lsis
3521; LY333531/LY379196; a isochinoline compound; a farnesyl
transferase inhibitor; PD184352 or QAN697, or AT7519; k) a compound
targeting, decreasing or inhibiting the activity of a
protein-tyrosine kinase, such as imatinib mesylate or a tyrphostin,
such as Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213;
Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin
B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556,
AG957 and adaphostin
(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl
ester; NSC 680410, adaphostin); 1) a compound targeting,
decreasing, or inhibiting the activity of the epidermal growth
factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3,
ErbB4 as homo- or heterodimers) and their mutants, such as CP
358774, ZD 1839, ZM 105180; trastuzumab, cetuximab, getfitinib,
erlotinib, OSI-774, Cl-1033, EKB-569, GW-2016, antibodies E1.1,
E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3, and
7H-pyrrolo-[2,3-d]pyrimidine derivatives; and m) a compound
targeting, decreasing, or inhibiting the activity of the c-Met
receptor.
Exemplary compounds that target, decrease, or inhibit the activity
of a protein or lipid phosphatase include inhibitors of phosphatase
1, phosphatase 2A, or CDC25, such as okadaic acid or a derivative
thereof.
Further anti-angiogenic compounds include compounds having another
mechanism for their activity unrelated to protein or lipid kinase
inhibition, e.g., thalidomide and TNP-470.
Additional, nonlimiting, exemplary chemotherapeutic compounds, one
or more of which may be used in combination with a present BET
bromodomain inhibitor, include: daunorubicin, adriamycin, Ara-C,
VP-16, teniposide, mitoxantrone, idarubicin, carboplatinum, PKC412,
6-mercaptopurine (6-MP), fludarabine phosphate, octreotide, SOM230,
FTY720, 6-thioguanine, cladribine, 6-mercaptopurine, pentostatin,
hydroxyurea, 2-hydroxy-1H-isoindole-1,3-dione derivatives,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a
pharmaceutically acceptable salt thereof,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate,
angiostatin, endostain, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon; FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-a-epihydrocotisol, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, and
siRNA.
Other examples of second therapeutic agents, one or more of which a
present BET bromodomain inhibitor also can be combined, include,
but are not limited to: a treatment for Alzheimer's Disease, such
as donepezil and rivastigmine; a treatment for Parkinson's Disease,
such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,
bromocriptine, pergolide, trihexephendyl, and amantadine; an agent
for treating multiple sclerosis (MS) such as beta interferon (e.g.,
AVONEX.RTM. and REBIF.RTM.), glatiramer acetate, and mitoxantrone;
a treatment for asthma, such as albuterol and montelukast; an agent
for treating schizophrenia, such as zyprexa, risperdal, seroquel,
and haloperidol; an anti-inflammatory agent, such as a
corticosteroid, a TNF blocker, IL-1 RA, azathioprine,
cyclophosphamide, and sulfasalazine; an immunomodulatory agent,
including immunosuppressive agents, such as cyclosporin,
tacrolimus, rapamycin, mycophenolate mofetil, an interferon, a
corticosteroid, cyclophosphamide, azathioprine, and sulfasalazine;
a neurotrophic factor, such as an acetylcholinesterase inhibitor,
an MAO inhibitor, an interferon, an anti-convulsant, an ion channel
blocker, riluzole, or an anti-Parkinson's agent; an agent for
treating cardiovascular disease, such as a beta-blocker, an ACE
inhibitor, a diuretic, a nitrate, a calcium channel blocker, or a
statin; an agent for treating liver disease, such as a
corticosteroid, cholestyramine, an interferon, and an anti-viral
agent; an agent for treating blood disorders, such as a
corticosteroid, an anti-leukemic agent, or a growth factor; or an
agent for treating immunodeficiency disorders, such as gamma
globulin.
The above-mentioned second therapeutically active agents, one or
more of which can be used in combination with a BET bromodomain
inhibitor of structural formula (I), are prepared and administered
as described in the art.
The compounds of the present invention typically are administered
in admixture with a pharmaceutical carrier selected with regard to
the intended route of administration and standard pharmaceutical
practice. Pharmaceutical compositions for use in accordance with
the present invention are formulated in a conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries that facilitate processing of compounds
of structural formula (I).
These pharmaceutical compositions can be manufactured, for example,
by conventional mixing, dissolving, granulating, dragee-making,
emulsifying, encapsulating, entrapping, or lyophilizing processes.
Proper formulation is dependent upon the route of administration
chosen. When a therapeutically effective amount of the compound of
structural formula (I) is administered orally, the composition
typically is in the form of a tablet, capsule, powder, solution, or
elixir. When administered in tablet form, the composition
additionally can contain a solid carrier, such as a gelatin or an
adjuvant. The tablet, capsule, and powder contain about 0.01% to
about 95%, and preferably from about 1% to about 50%, of a compound
of structural formula (I). When administered in liquid form, a
liquid carrier, such as water, petroleum, or oils of animal or
plant origin, can be added. The liquid form of the composition can
further contain physiological saline solution, dextrose or other
saccharide solutions, or glycols. When administered in liquid form,
the composition contains about 0.1% to about 90%, and preferably
about 1% to about 50%, by weight, of a compound of structural
formula (I).
When a therapeutically effective amount of a compound of structural
formula (I) is administered by intravenous, cutaneous, or
subcutaneous injection, the composition is in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable solutions, having due
regard to pH, isotonicity, stability, and the like, is within the
skill in the art. A preferred composition for intravenous,
cutaneous, or subcutaneous injection typically contains, an
isotonic vehicle.
Compounds of structural formula (I) can be readily combined with
pharmaceutically acceptable carriers well-known in the art. Such
carriers enable the active agents to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can be obtained
by adding the compound of structural formula (I) to a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients include, for example, fillers and cellulose
preparations. If desired, disintegrating agents can be added.
A compound of structural formula (I) can be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampules or in multidose containers, with
an added preservative. The compositions can take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing,
and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include
aqueous solutions of the active agent in water-soluble form.
Additionally, suspensions of a compound of structural formula (I)
can be prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils or synthetic
fatty acid esters. Aqueous injection suspensions can contain
substances which increase the viscosity of the suspension.
Optionally, the suspension also can contain suitable stabilizers or
agents that increase the solubility of the compounds and allow for
the preparation of highly concentrated solutions. Alternatively, a
present composition can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
A compound of structural formula (I) also can be formulated in
rectal compositions, such as suppositories or retention enemas,
e.g., containing conventional suppository bases. In addition to the
formulations described previously, the compound of structural
formula (I) also can be formulated as a depot preparation. Such
long-acting formulations can be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds of structural formula
(I) can be formulated with suitable polymeric or hydrophobic
materials (for example, as an emulsion in an acceptable oil) or ion
exchange resins.
In particular, the compounds of structural formula (I) can be
administered orally, buccally, or sublingually in the form of
tablets containing excipients, such as starch or lactose, or in
capsules or ovules, either alone or in admixture with excipients,
or in the form of elixirs or suspensions containing flavoring or
coloring agents. Such liquid preparations can be prepared with
pharmaceutically acceptable additives, such as suspending agents.
The compounds of structural formula (I) also can be injected
parenterally, for example, intravenously, intramuscularly,
subcutaneously, or intracoronarily. For parenteral administration,
the present BET bromodomain inhibitors are best used in the form of
a sterile aqueous solution which can contain other substances, for
example, salts or monosaccharides, such as mannitol or glucose, to
make the solution isotonic with blood.
As an additional embodiment, the present invention includes kits
which comprise one or more compounds or compositions packaged in a
manner that facilitates their use to practice methods of the
invention. In one simple embodiment, the kit includes a compound or
composition described herein as useful for practice of a method
(e.g., a composition comprising a compound of structural formula
(I) and an optional second therapeutic agent), packaged in a
container, such as a sealed bottle or vessel, with a label affixed
to the container or included in the kit that describes use of the
compound or composition to practice the method of the invention.
Preferably, the compound or composition is packaged in a unit
dosage form. The kit further can include a device suitable for
administering the composition according to the intended route of
administration.
Prior BET bromodomain inhibitors possessed properties that hindered
their development as therapeutic agents. In accordance with an
important feature of the present invention, compounds of structural
formula (I) were synthesized and evaluated as inhibitors for BET
bromodomains. For example, compounds of the present invention
typically have a bonding affinity (IC.sub.50) to BET bromodomains
of less than 100 .mu.M, less than 50 .mu.M, less than 25 .mu.M, and
less than 5 .mu.M.
Synthesis of Compounds
Compounds of the present invention and were prepared as follows.
The following synthetic schemes are representative of the reactions
used to synthesize compounds of structural formula (I).
Modifications and alternate schemes to prepare BET bromodomain
inhibitors of the invention are readily within the capabilities of
persons skilled in the art.
Solvents and reagents were obtained commercially and used without
further purification. Chemical shifts (.delta.) of NMR spectra are
reported as .delta. values (ppm) downfield relative to an internal
standard, with multiplicities reported in the usual manner.
Unless otherwise stated all temperatures are in degrees
Celsius.
In the synthetic methods, the examples, and throughout the
specification, the abbreviations have the following meanings
TABLE-US-00001 DMF dimethylformamide min minutes
CH.sub.2Cl.sub.2/DCM methylene chloride MeOH methanol
Na.sub.2SO.sub.4 sodium sulfate AcOH acetic acid MS mass
spectrometry Na.sub.2CO.sub.3 sodium carbonate Br.sub.2 bromine h
hours CH.sub.3I/MeI methyl iodide CHCl.sub.3 chloroform N.sub.2
nitrogen gas H.sub.2N--NH.sub.2 hydrazine H.sub.2 hydrogen gas
POCl.sub.3 phosphorous oxytrichloride EtOAc ethyl acetate KOAc
potassium acetate NaOAc sodium acetate Na.sub.2SO.sub.3 sodium
sulfite Na.sub.2SO.sub.4 sodium sulfate NaHCO.sub.3 sodium
bicarbonate HCl hydrochloric acid g gram mol mole mmol millimole mL
milliliter KOH potassium hydroxide NH.sub.2OH.cndot.HCl
hydroxylamine hydrochloride CD.sub.3OD/MeOD deuterated methanol M
molar N normal RT/rt room temperature DME 1,2-dimethoxyethane NMR
nuclear magnetic resonance spectrometry THF tetrahydrofuran
NEt.sub.3 triethylamine CDCl.sub.3 deuterated chloroform Hz Hertz
Ar aryl H.sub.2O water EtOH ethanol DMAP 4-dimethylaminopyridine
K.sub.2CO.sub.3 potassium carbonate NIS N-iodosuccinimide NBS
N-bromosuccinimide NaH sodium hydride Zn zinc NH.sub.4Cl ammonium
chloride Pd(dppf)Cl.sub.2
[1,1'-bis(diphenylphosphino)ferrocene]dichloro palladium (II)
CF.sub.3CO.sub.2H/TFA trifluoroacetic acid EtN(iPr).sub.2/DIPEA
diisopropylethylamine PyHBr.sub.3 pyridinium tribromide NH.sub.3
ammonia Pd/C palladium on carbon
(PPh.sub.3).sub.4Pd/Pd(PPh.sub.3).sub.4
tetrakis(triphenylphosphine)pallad- ium(0) n-BuLi n-butyl lithium
PCC pyridinium chlorochromate Et.sub.2O diethyl ether (PhO).sub.2
PO--N.sub.3 (DPPA) diphenyl phosphorazidate CuI cupric iodide
HCO.sub.2NH.sub.4 ammonium formate H.sub.2N-CHO formamide
All final compounds are in trifluoroacetate salt form. The cations
are not drawn in the following structures.
1. Synthesis of General Intermediates: RX3 or RX103
##STR00100##
An aqueous solution (20 mL) of 1,4-cyclohexanediol (17.5 g, 150
mmol) and KOH (9.3 g, 170 mmol) was heated to reflux for one hour.
After cooling to room temperature, water was removed under reduced
pressure, then CH.sub.3I (32.0 g, 230 mmol) was added. After 24
hours stirring at room temperature, the reaction mixture was
quenched with 100 mL water, and extracted with CHCl.sub.3 (100
mL.times.3). The combined organic fraction was dried, then purified
in flash column chromatography (washed out at ethyl acetate:
hexane=1:1) to give 7.14 g (36%) pale yellow liquid as the titled
compound (Known compound, ACS Registry No. 18068-06-9).
##STR00101##
4-methoxycyclohexanone
4-Methoxycyclohexanol (41.9 g, 322 mmol) was dissolved in DCM (360
mL), and added slowly into a pyridinium chlorochromate (138.8 g,
644 mmol) DCM solution (720 mL). The resulting mixture was stirred
for 4 hours under N.sub.2 protection. Pyridinium chlorochromate as
filtered with H type silica gel, and the filtrate was concentrated
and purified with flash column (eluent EtOAc:Hexane=1:1) to give
38.4 g (93%) tilted compound as a pale yellow oil. 1HNMR (300 MHz,
CDCl.sub.3) .delta. 3.61 (t, 1H, J=2.4 Hz), 3.40 (s, 3H), 2.56 (m,
2H), 2.26 (m, 2H), 2.10 (m, 2H), 1.96 (m, 2H).
##STR00102##
4-hydrazinylpyridin-2(1H)-one
4-Hydrazinylpyridin-2(1H)-one (4.97 g, 44.7 mmol) was added slowly
into a 2-methoxyethanol solution of (100 mL) H.sub.2N--NH.sub.2
(9.19 g, 290 mmol). The mixture was heated to reflux for 24 hours,
after which the solvent was removed and 4.57 g (81.6%) titled
compound was given through recrystallization in ethanol. .sup.1HNMR
(300 MHz, D2O) .delta. 7.67 (s, 1H), 7.24 (d, 2H, J=7.2 Hz), 630
(s, 1H), 6.04 (d, 2H, J=7.2 Hz), 5.73 (s, 1H), 3.62 (s, 2H).
##STR00103##
4-Hydrazinyl-6-methylpyridin-2(1H)-one
4-Hydroxy-6-methylpyridin-2(1H)-one (25 g, 200 mmol) and hydrazine
monohydrate (65 g, 1299 mmol) mixture in 2-methoxyethanol (500 mL)
was heated to reflux for 24 hours. After cooled to room
temperature, the product was crystallized in ethanol (22.2 g,
79.8%). .sup.1HNMR (300 MHz, DMSO-d.sub.6) .delta. 10.22 (br, 1H),
7.40 (s, 1H), 5.41 (s, 1H), 5.24 (s, 1H), 4.04 (d, 2H, J=1.5 Hz),
1.99 (s, 3H).
##STR00104##
4-(2-4-methoxycyclohexylidene)hydrazinyl)pyridine-2(1H)-one
4-Hydrazinylpyridin-2(1H)-one (2.07 g, 16.5 mmol) was suspended in
4-methoxycyclohexanone (2.33 g, 18.2 mmol) solution in absolute
ethanol (100 mL). After being heated to reflux for 2 hours, the
reaction mixture was concentrated to half of its original volume.
The resulting precipitates were filtered and dried to give 3.02 g
(77.0%) colorless solid. .sup.1HNMR (300 MHz, DMSO-d.sub.6) .delta.
10.50 (s, 1H), 9.28 (s, 1H), 7.07 (d, 1H, J=7.2 Hz), 6.01 (d, 1H,
J=5.7 Hz), 5.67 (d, 1H, J=2.1 Hz), 3.45 (m, 1H), 3.28 (s, 3H), 2.35
(m, 2H), 2.20 (m, 2H), 1.86 (m, 2H), 1.62 (m, 2H).
##STR00105##
4-(2-(4-Methoxycyclohexylidene)hydrazinyl)-6-methylpyridin-2(1H)-one
4-Hydrazinyl-6-methylpyridin-2(1H)-one (16.33 g, 117 mmol) was
suspended in an ethanol solution of 4-methoxycyclohexanone (16.5 g,
129 mmol). The mixture was heated to reflux for 2 hours, and
concentrated to half of its volume. Filtered the precipitate and
evaporated the filtrate to give colorless powder (30 g) which was
used in next step without further purification. .sup.1HNMR (300
MHz, DMSO-d.sub.6) .delta. 6.12 (s, 1H), 5.90 (s, 1H), 3.40 (s,
3H), 2.48-3.40 (m, 2H), 2.28-2.41 (m, 2H), 2.22 (s, 3H), 1.88-2.03
(m, 2H), 1.71-1.78 (m, 2H).
##STR00106##
8-Methoxy-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one
4-(2-(4-Methoxycyclohexylidene) hydrazinyl)pyridin-2(1H)-one (20.1
g, 85.4 mmol) was suspended in 400 mL diphenyl ether. The mixture
was heated to reflux under N.sub.2 protection for 30 minutes. After
cooling to room temperature, 10% Pd/C (6 g) was added and the
mixture was heated to reflux again for 75 minutes. Then, hexane
(800 mL) was added to the cooled mixture. The resulting
precipitates were filtered and taken up into boiling AcOH (1100
mL), followed by filtering again to remove Pd--C. The filtrate was
concentrated to give yellow solid, which was boiled in 8 mL
ethanol. Then, the solid was filtered to give 9 g (50%) pale yellow
solid as the titled compound. .sup.1HNMR (300 MHz, DMSO-d.sub.6)
.delta. 11.54 (s, 1H), 11.03 (s, 1H), 7.60 (d, 1H, J-2.1 Hz), 7.37
(d, 1H, J=8.7 Hz), 7.25 (m, 1H), 6.90 (dd, 1H, J.sub.1=2.7 Hz,
J.sub.2=8.7 Hz), 6.46 (d, 1H, J=7.2 Hz), 3.81 (s, 3H).
##STR00107##
8-Methoxy-3-methyl-5H-pyrido[4, 3-b]indol-1-ol
4-(2-(4-methoxycyclohexylidene)hydrazinyl)-6-methylpyridin-2(1H)-one
(1.99 g, 7.99 mmol) was refluxed in 60 mL diphenyl ether for 30
minutes, after cooled to room temperature, Pd--C (6.1 g, 0.57 mmol)
was added and heat for additional 1.25 hours. Let it cooled, and
precipitate with hexane (80 mL). The filter was taken up into hot
AcOH (110 mL) and Pd--C was removed by filtration. Then evaporated
AcOH and wash the crude product by having it boiled in MeOH (16
mL). The solid was collected and put into next step without further
purification (1 g, 54.8%).
##STR00108##
1-chloro-8-methoxy-5H-pyridol[4, 3-1)] indole
POCl.sub.3 (20 mL) and 8-methoxy-5H-pyridol[4,3-b]indol-1-ol were
refluxed for 24 hours followed by removal of POCl.sub.3 under
reduced pressure. The residue was refluxed with HCl for additional
1 hour. After cooling, the mixture was neutralized with ammonium
hydroxide, the precipitate was filtered and purified with flash
column chromatography (EtOAc:Hexane=1:1 as eluent) to give 0.44 g
(64.5%) titled compound as a colorless powder. .sup.1HNMR (300 MHz,
MeOD-d.sub.4) .delta. 8.26 (d, 1H, J-6.3 Hz), 7.94 (d, 1H, J=2.1
Hz), 7.61 (m, 2H), 7.31 (dd, 1H, J.sub.1=2.1 Hz, J.sub.2=8.7 Hz),
3.96 (s, 3H).
##STR00109##
7-bromo-1-chloro-8-methoxy-5H-pyrido[4,3-b]indole
1-Chloro-8-methoxy-5H-pyrido[4,3-b]indole (377 mg, 1.6 mmol) and
NaOAc (197 mg, 2.4 mmol) were dissolved in AcOH (40 mL). Then
bromine (389 mg, 2.4 mmol) was added dropwisely. After stirring at
room temperature for overnight, the reaction was quenched with
Na.sub.2SO.sub.3 solution. AcOH was then removed under reduced
pressure and the resulting aqueous phase was extracted with EtOAc.
The combined organic fraction was concentrated and purified with
prep-HPLC to give 157 mg (31.1%) colorless powder. .sup.1HNMR (300
MHz, MeOD-d.sub.4) .delta. 8.21 (d, 1H, J=5.7 Hz), 8.00 (s, 1H),
7.84 (s, 1H), 7.50 (d, J=6.0 Hz), 4.03 (s, 3H).
##STR00110##
4-(1-chloro-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylisoxazole
(RX3)
7-Bromo-1-chloro-8-methoxy-5H-pyrido[4,3-b]indole (157 mg, 0.5
mmol), 3,5-dimethylisoxazole-4-boronic acid pinacol ester (655 mg,
2.0 mmol), and K.sub.2CO.sub.3 (345 mg, 2.5 mmol) were dissolved in
DME/H.sub.2O (50 mL/25 mL) system. Then vacuumed, and refilled with
N.sub.2. After that, tetraki(triphenylphosphine)palladium (0) was
added, followed by vacuuming and refilling with N.sub.2. The
reaction mixture was heated to reflux for overnight, when cooled to
room temperature, it was extracted with EtOAc, and the combined
organic fractions were concentrated before purification in
prep-HPLC. 57 mg (34.6%) of the titled compound was obtained after
being lyophilized for 24 hours as a pale yellow powder. .sup.1HNMR
(300 MHz, MeOD-d.sub.4) .delta. 8.26 (d, 1H, J=6.0 Hz), 8.09 (s,
1H), 7.60 (d, 1H, J=6.3 Hz), 7.49 (s, 1H), 3.98 (s, 3H), 2.63 (s,
3H), 2.20 (s, 3H). ESIMS m/z [M+H].sup.+ calculated=328.77.
found=328.83.
##STR00111##
4-(1-chloro-8-methoxy-3-methyl-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyliso-
xazole
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.03 (s, 1H), 7.44 (s,
1H), 7.42 (s, 1H), 3.97 (s, 3H), 2.69 (s, 3H), 2.36 (s, 3H), 2.19
(s, 3H). ESIMS m/z [M+H].sup.+ calcd.=342.80. found=342.58.
2. General Methods for Syntheses of Five-Membered Heterocyclic
Containing Pinacol Boronates
##STR00112##
R.sub.1, R.sub.2=methyl, ethyl, propyl, isopropyl, cyclopropyl,
cyclobutyl, phenyl, other alkyl, heteroaryl, or aryl.
Y=N
Z=NH, O, N-alkyl
X=Br, I
Example 1
##STR00113##
Example 2
##STR00114##
3-ethyl-5-methylisoxazole. NH.sub.2OH.HCl (0.542 g, 7.8 mmol) was
dissolved in MeOH/H.sub.2O (10 mL/20 ml), followed by addition of a
Na.sub.2CO.sub.3 (0.413 g, 3.9 mmol). When bubbles were absent,
hexane-2,4-dione was added dropwisely. The mixture was heated at
reflux overnight. After cooling to room temperature, the mixture
was extracted with Et.sub.2O (40 mL.times.2), and dried over
Na.sub.2SO.sub.4 anhydrous. Removal of solvent to give 0.559 g
(Yield: 57.3%) light yellow liquid. .sup.1HNMR (300 MHz,
CDCl.sub.3), .delta. 5.82 (s, 1H), 2.74 (q, J=7.5 Hz, 2H), 2.28 (s,
3H), 1.28 (q, J=7.5 Hz, 3H).
##STR00115##
5-ethyl-3-methylisoxazole. .sup.1HNMR (300 MHz, CDCl.sub.3),
.delta. 5.85 (s, 1H), 2.66 (q, J=7.5 Hz, 2H), 2.40 (s, 3H), 1.28
(q, J=7.5 Hz, 3H).
##STR00116##
4-bromo-3-ethyl-5-methylisoxazole. A mixture of
3-ethyl-5-methylisoxazole (0.22 g, 2.01 mmol) and NBS (0.39 g, 2.21
mmol) in DMF (5 mL) was stirred at room temperature overnight.
Then, the mixture was poured into ethyl acetate (20 mL) and
extracted with water (20 mL.times.5). The combined organic phase
was washed with saturated saline (20 mL), and dried over
Na.sub.2SO.sub.4 anhydrous. The 4-bromo-3-ethyl-5-methylisoxazole
was purified with silica gel flash column (washed out at ethyl
acetate:hexanes=1.30) to give 0.338 g (Yield: 84.7%) light yellow
liquid. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 2.77 (q, J=7.5
Hz, 2H), 2.28 (s, 3H), 130 (t, J=7.5 Hz, 3H).
##STR00117##
4-bromo-5-ethyl-3-methylisoxazole. .sup.1HNMR (300 MHz,
CDCl.sub.3), .delta. 2.67 (q, J=7.5 Hz, 2H), 2.41 (s, 3H), 1.30 (t,
J=7.5 Hz, 3H).
##STR00118##
3-ethyl-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole-
. Synthesis was performed using the general methods for syntheses
of pinacol boronates. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta.
2.93 (q, J=7.5 Hz, 2H), 2.35 (s, 3H), 1.32 (s, 12H), 1.26 (m,
3H).
##STR00119##
5-ethyl-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole-
. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 2.77 (q, J=7.5 Hz, 2H),
2.53 (s, 3H), 1.32 (s, 12H), 1.26 (m, 3H).
##STR00120##
3,5-diethyl-1H-pyrazole. A mixture of heptane-3,5-dione (1.128 g,
8.8 mmol) and hydrazine hydrate (0.44 g, 8.9 mmol) was heated to
reflux for 1 hour. Then, the mixture was extracted with ethyl
acetate and dried over Na.sub.2SO.sub.4 anhydrous. Removal of
solvent gave 0.96 g (Yield: 87.8%) bright yellow oil. .sup.1HNMR
(300 MHz, CDCl.sub.3), .delta. 5.89 (s, 1H), 2.67 (q, J=7.5 Hz,
6H).
##STR00121##
3,5-diethyl-4-iodo-1H-pyrazole. .sup.1HNMR (300 MHz, CDCl.sub.3),
.delta. 2.65 (q, J=7.5 Hz, 4H), 1.27 (t, J=7.5 Hz, 6H).
##STR00122##
tert-butyl 3,5-diethyl-4-iodo-1H-pyrazole-1-carboxylate. A mixture
of 3,5-diethyl-4-iodo-1H-pyrazole and (Boc).sub.2O was dissolved in
THF and stirred at room temperature for 1 hour. The product then
was purified with silica gel column (ethyl acetate:hexanes=1:3).
.sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 3.01 (q, J=7.5 Hz, 2H),
2.65 (q, J=7.5 Hz, 2H), 1.66 (s, 9H), 1.28 (t, J=7.5 Hz, 3H), 1.18
(t, J=7.5 Hz, 3H).
##STR00123##
tert-butyl
3,5-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-
-carboxylate. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta.3.15 (q,
J=7.5 Hz, 2H), 2.74 (q, J=7.5 Hz, 2H), 1.61 (s, 9H), 1.27 (s, 12H),
1.17 (m, 6H).
##STR00124##
5-cyclopropyl-3-methyl-1H-pyrazole. .sup.1HNMR (300 MHz,
CDCl.sub.3) .delta. 9.01 (br, 1H), 5.72 (s, 1H), 2.27 (s, 3H), 1.90
(m, 1H), 0.92 (m, 2H), 0.70 (m, 2H).
##STR00125##
5-cyclopropyl-4-iodo-3-methyl-1H-pyrazole. .sup.1HNMR (300 MHz,
CDCl.sub.3) .delta. 2.23 (s, 3H), 1.83 (m, 1H), 0.95 (m, 2H), 0.80
(m, 2H)
##STR00126##
tert-butyl 5-cyclopropyl-4-iodo-3-methyl-1H-pyrazole-1-carboxylate.
.sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 2.52 (s, 3H), 1.82 (m,
1H), 1.61 (s, 9H), 0.98 (m, 2H), 0.90 (m, 2H).
##STR00127##
tert-butyl
5-cyclopropyl-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
-pyrazole-1-carboxylate. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta.
2.65 (s, 3H), 2.28 (m, 1H), 1.62 (s, 9H), 1.33 (s, 12H), 0.99 (m,
2H), 0.88 (m, 2H).
##STR00128##
4-bromo-5-methyl-3-phenyl-1H-pyrazole. .sup.1HNMR (300 MHz,
CDCl.sub.3), .delta. 10.05 (br, 1H), 7.80 (m, 2H), 7.46 (m, 3H),
2.36 (s, 3H).
##STR00129##
tert-butyl 4-bromo-5-methyl-3-phenyl-1H-pyrazole-1-carboxylate.
.sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 7.94 (m, 2H), 7.45 (m,
3H), 2.62 (s, 3H), 1.69 (s, 9H).
##STR00130##
tert-butyl
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole--
1-carboxylate. .sup.1HNMR (300 MHz, CDCl.sub.3), .delta. 7.81 (m,
2H), 7.37 (m, 3H), 2.76 (s, 3H), 1.67 (s, 9H), 1.32 (s, 12H).
3. Synthesis of Final Compounds from the General Intermediates
3.1 Reduction:
##STR00131##
4-(8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylisoxazole. 10%
Pd--C (5 mg) was suspended in an MeOH solution of
4-(1-chloro-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylisoxazole
(15 mg, 0.046 mmol). The reaction proceeded for 26 hours under a
H.sub.2 balloon at room temperature. Pd--C was filtered and the
filtrate was purified in semi-prep HPLC to give 4 mg (30%)
colorless powder after being lyophilized for 24 hours. .sup.1HNMR
(300 MHz, MeOD-d.sub.4) .delta. 9.61 (s, 1H), 8.54 (d, 1H, J=6.9
Hz), 8.11 (s, 1H), 7.97 (s, 1H), 4.00 (s, 3H), 2.36 (s, 3H), 2.19
(s, 3H). ESIMS m/z [M+H].sup.+ calculated=294.33. found=294.75.
3.2 General method for Suzuki Coupling:
##STR00132##
RX3 (20 mg, 0.06 mmol), the boronic acid or boronic acid pinacol
ester (4 equivalent), and K.sub.2CO.sub.3 (41.5 mg, 0.3 mmol) was
stirred in 15 mL H.sub.2O/DME (1:2). The mixture was vacuumed and
Pd(PPh.sub.3).sub.4 was added before heating to reflux under
N.sub.2 protection. After reflux overnight, the reaction was cooled
to room temperature, and extracted with ethyl acetate. After
removing the organic phase, the residue was purified using RP-HPLC
and a colorless powder was obtained after overnight
lyophilization.
##STR00133##
4,4'-(8-methoxy-5H-pyrido[4,3-b]indole-1,7-diyl)bis(3,5-dimethylisoxazole-
). .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.63 (d, 1H, J=6.6
Hz), 8.04 (d, 1H, J=6.6 Hz), 7.67 (s, 1H), 7.02 (s, 1H), 3.78 (s,
3H), 2.52 (s, 3H), 2.34 (s, 3H), 2.28 (s, 3H), 2.17 (s, 3H). ESIMS
m/z [M+H].sup.+ calculated=389.43. found=389.50.
##STR00134##
4-(8-methoxy-1-(1H-pyrazol-4-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyl-
isoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.48 (s, 2H),
8.45 (d, 1H, J=6.9 Hz), 7.89 (d, 1H, J=6.9 Hz), 7.63 (s, 1H), 7.61
(s, 1H), 3.82 (s, 3H), 2.35 (s, 3H), 2.17 (s, 3H). ESIMS m/z
[M+H].sup.+ calculated=360.39. found=361.17.
##STR00135##
4-(8-methoxy-1-phenyl-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylisoxazole.
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.53 (d, 1H, J=6.9 Hz),
7.98 (m, 3H), 7.86 (m, 3H), 7.61 (s, 1H), 7.17 (s, 1H), 3.63 (s,
3H), 2.33 (s, 3H), 2.15 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=370.42. found=370.42.
##STR00136##
4-(8-methoxy-1-(pyridin-3-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyliso-
xazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 9.21 (br, 1H),
9.06 (br, 1H), 8.61 (d, 1H, J=6.9 Hz), 8.49 (d, 1H, J=7.8 Hz), 8.03
(d, 1H, J=6.6 Hz), 7.93 (br, 1H), 7.65 (s, 1H), 7.07 (s, 1H), 3.67
(s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=371.41. found=371.75.
##STR00137##
4-(1-(3-chlorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.56 (d, 1H,
J=6.9 Hz), 8.09 (d, 1H, J=1.2 Hz), 8.02 (d, 1H, J=6.9 Hz),
7.85-7.93 (m, 3H), 7.65 (s, 1H), 7.20 (s, 1H), 3.69 (s, 3H), 2.34
(s, 3H), 2.16 (s, 3H). ESIMS m/z [M+H].sup.+ calculated=404.87.
found=405.00.
##STR00138##
4-(1-(2-chlorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.62 (d, 1H,
J=6.9 Hz), 8.04 (d, 1H, J=6.6 Hz), 7.89 (m, 3H), 7.80 (m, 1H), 7.63
(s, 1H), 6.64 (s, 1H), 3.56 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H).
ESIMS m/z [M+H].sup.+ calculated=404.87. found=404.92.
##STR00139##
4-(1-(4-chlorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.54 (d, 1H,
J=6.9 Hz), 8.00 (d, 2H, J=8.7 Hz), 7.98 (d, 1H, J=6.6 Hz), 7.88 (d,
2H, J=8.7 Hz), 7.62 (s, 1H), 7.19 (s, 1H), 3.69 (s, 3H), 2.33 (s,
3H), 2.16 (s, 3H). ESIMS m/z [M+H].sup.+ calculated=404.87.
found=405.33.
##STR00140##
4-(1-([1,1'-biphenyl]-3-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dim-
ethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.56 (d,
1H, J=6.9 Hz), 8.29 (s, 1H), 8.15 (m, 1H), 7.98 (m, 3H), 7.81 (d,
2H, J=7.2 Hz), 7.62 (s, 1H), 7.50 (m, 3H), 7.26 (s, 1H), 3.50 (s,
3H), 2.32 (s, 3H), 2.14 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=446.52. found=446.75.
##STR00141##
4-(1-([1,1'-biphenyl]-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dim-
ethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.55 (d,
1H, J=6.9 Hz), 8.13 (d, 2H, J=8.7 Hz), 8.08 (d, 2H, J=8.4 Hz), 7.99
(d, 1H, J=6.6 Hz), 7.83 (dd, 2H, J.sub.1=7.8 Hz, J.sub.2=1.2 Hz),
7.63 (s, 1H), 7.48-7.59 (m, 3H), 7.31 (s, 1H), 3.66 (s, 3H), 2.34
(s, 3H), 2.16 (s, 3H). ESIMS m/z [M+H].sup.+ calculated=446.52.
found=446.92.
##STR00142##
4-(8-methoxy-1-(naphthalen-1-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyl-
isoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.65 (d, 1H,
J=6.9 Hz), 8.40 (d, 1H, J=8.1 Hz), 8.22 (d, 1H, J=8.4 Hz), 8.09 (d,
1H, J=6.9 Hz), 7.93 (m, 2H), 7.70 (m, 1H), 7.59 (s, 1H), 7.54 (m,
1H), 6.10 (s, 1H), 3.11 (s, 3H), 2.27 (s, 1H), 2.07 (s, 1H). ESIMS
m/z [M+H].sup.+ calculated=420.48. found=420.75.
##STR00143##
4-(8-methoxy-1-(naphthalen-2-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyl-
isoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.57 (d, 1H,
J=6.9 Hz), 8.36 (d, 1H, J=8.4 Hz), 8.17 (dd, 2H, J.sub.1=6.9 Hz,
J.sub.2=1.5 Hz), 8.04 (dd, 1H, J.sub.1=8.4 Hz, J.sub.2=1.8 Hz),
8.00 (d, 1H, J=6.6 Hz), 7.75 (m, 2H), 7.63 (s, 1H), 7.26 (s, 1H),
3.48 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=420.48. found=420.92.
##STR00144##
4-(1-(2-fluorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.61 (d, 1H,
J=6.6 Hz), 8.03 (d, 1H, J=6.9 Hz), 7.94 (m, 2H), 7.66 (m, 3H), 6.96
(s, 1H), 3.62 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESIMS m/z
[M+H].sup.+ calculated=388.41. found=388.75.
##STR00145##
4-(1-(3-fluorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.55 (d, 1H,
J=6.9 Hz), 8.00 (d, 1H, J=6.9 Hz), 7.85 (m, 3H), 7.67 (m, 1H), 7.63
(s, 1H), 7.18 (s, 1H), 3.67 (s, 3H), 2.34 (s, 3H), 2.16 (s, 3H).
ESIMS m/z [M+H].sup.+ calculated=388.41. found=388.50.
##STR00146##
4-(1-(4-fluorophenyl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyli-
soxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.53 (d, 1H,
J=6.9 Hz), 8.05 (m, 2H), 7.98 (d, 1H, J=6.6 Hz), 7.61 (m, 2H), 7.62
(s, 1H), 7.18 (s, 1H), 3.68 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H).
ESIMS m/z [M+H].sup.+ calculated=388.41. found=389.08.
##STR00147##
4-(8-methoxy-1-(pyridin-4-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethyliso-
xazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 9.07 (d, 2H,
J=5.7 Hz), 8.62 (d, 1H, J=6.9 Hz), 8.06 (m, 3H), 7.65 (s, 1H), 7.12
(s, 1H), 3.67 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESIMS m/z
[M+H].sup.+ calculated=371.41. found=372.25.
##STR00148##
4-(1-(1H-indol-5-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylis-
oxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.47 (d, 1H,
J=6.6 Hz), 8.25 (d, 1H, J=1.5 Hz), 7.91 (d, 1H, J=6.6 Hz), 7.84 (d,
1H, J=8.4 Hz), 7.70 (dd, 1H, J.sub.1=8.4 Hz, J.sub.2=1.8 Hz), 7.60
(s, 1H), 7.53 (d, 1H, J=3.0 Hz), 7.42 (s, 1H), 6.76 (d, 1H, J=3.3
Hz), 3.56 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESIMS m/z
[M+H].sup.+ calculated=409.46. found=409.67.
##STR00149##
4-(1-(1H-indol-3-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylis-
oxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.46 (d, 1H,
J=6.9 Hz), 8.13 (s, 1H), 7.89 (d, 1H, J=6.6 Hz), 7.72 (d, 1H, J=8.7
Hz), 7.59 (s, 1H), 7.40 (m, 2H), 7.25 (m, 1H), 7.01 (s, 1H), 3.30
(s, 3H), 2.33 (s, 3H), 2.14 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=409.46. found=409.67.
##STR00150##
4-(1-(3-cyclopropyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl-
)-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta.
8.51 (d, 1H, J=6.9 Hz), 8.17 (s, 1H), 7.94 (d, 1H, J=6.9 Hz), 7.62
(s, 1H), 7.31 (s, 1H), 3.78 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H),
0.93 (m, 5H). ESIMS m/z [M+H].sup.+ calculated=400.45.
found=400.67.
##STR00151##
4-(1-(3-isopropyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)--
3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta.
8.52 (d, 1H, J=6.6 Hz), 8.11 (s, 1H), 7.96 (d, 1H, J=6.9 Hz), 7.62
(s, 1H), 7.01 (s, 1H), 3.70 (s, 3H), 2.34 (s, 3H), 2.16 (s, 3H),
1.34-1.22 (m, 7H). ESIMS m/z [M+H].sup.+ calculated=402.47.
found=402.92.
##STR00152##
4-(8-methoxy-1-(3-methyl-1H-pyrazol-4-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-
-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.50
(d, 1H, J=6.9 Hz), 8.22 (s, 1H), 7.93 (d, 1H, J=6.6 Hz), 7.62 (s,
1H), 7.23 (s, 1H), 3.76 (s, 3H), 2.40 (s, 3H), 2.34 (s, 3H), 2.17
(s, 3H). ESIMS m/z [M+H].sup.+ calculated=374.42. found=374.25.
##STR00153##
4-(1-(3,5-dimethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-
-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta.
8.54 (d, 1H, J=6.9 Hz), 7.95 (d, 1H, J=6.9 Hz), 7.63 (s, 1H), 7.02
(s, 1H), 3.74 (s, 3H), 2.34 (s, 3H), 2.29 (s, 6H), 2.16 (s, 3H).
ESIMS m/z [M+H].sup.+ calculated=388.44. found=388.42.
##STR00154##
4-(1-(3,5-diethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)--
3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta.
8.55 (d, 1H, J=6.9 Hz), 7.97 (d, 1H, J=6.9 Hz), 7.64 (s, 1H), 6.92
(s, 1H), 3.70 (s, 3H), 2.65 (m, 4H). 2.34 (s, 3H), 2.16 (s, 3H),
1.10 (t, 6H, J=7.5 Hz). ESIMS m/z [M+H].sup.+ calculated=416.50.
found=416.42.
##STR00155##
4-(1-(3-cyclopropyl-5-methyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]i-
ndol-7-yl)-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz,
MeOD-d.sub.4) .delta. 8.55 (d, 1H, J=6.9 Hz), 7.96 (d, 1H, J=6.6
Hz), 7.63 (s, 1H), 7.12 (s, 1H), 3.76 (s, 3H), 2.35 (s, 3H), 2.28
(s, 3H), 2.17 (s, 3H), 1.73 (m, 1H), 0.87 (m, 4H). ESIMS m/z
[M+H].sup.+ calculated=414.48. found=414.50.
##STR00156##
4-(8-methoxy-1-(1,3,5-trimethyl-1H-pyrazol-4-yl)-5H-pyrido[4,3-b]indol-7--
yl)-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4)
.delta. 8.54 (d, 1H, J=6.9 Hz), 7.95 (d, 1H, J=6.9 Hz), 7.63 (s,
1H), 7.04 (s, 1H), 3.97 (s, 3H), 3.75 (s, 3H), 2.34 (s, 3H), 2.32
(s, 3H), 2.21 (s, 3H), 2.16 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=402.47. found=402.75.
##STR00157##
4-(8-methoxy-1-(5-methyl-3-phenyl-1H-pyrazol-4-yl)-5H-pyrido[4,3-b]indol--
7-yl)-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4)
.delta. 8.54 (d, 1H, J=6.9 Hz), 7.98 (d, 1H, J=6.9 Hz), 7.57 (s,
1H), 7.34 (m, 2H), 7.25 (m, 3H), 6.98 (s, 1H), 3.70 (s, 3H), 2.33
(s, 3H), 2.30 (s, 3H), 2.12 (s, 3H). ESIMS m/z [M+H].sup.+
calculated=450.51. found=450.75.
##STR00158##
4-(1-(5-Cyclopropyl-1,3-dimethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-
-b]indol-7-yl)-3,5-dimethylisoxazole .sup.1HNMR (300 MHz,
MeOD-d.sub.4) .delta. 8.56 (d, 1H, J=6.9 Hz), 7.97 (d, 1H, J=6.9
Hz), 7.64 (s, 1H), 6.98 (s, 1H), 4.08 (s, 3H), 3.74 (s, 3H), 2.34
(s, 3H), 2.17 (s, 3H), 2.16 (s, 3H), 1.99 (m, 1H), 0.83 (m, 2H),
0.38 (m, 1H), 0.14 (m, 1H). ESIMS m/z [M+H].sup.+
calculated=428.51. found=428.42.
##STR00159##
4-(1-(3-Cyclopropyl-1,5-dimethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-
-b]indol-7-yl)-3,5-dimethylisoxazole .sup.1HNMR (300 MHz,
MeOD-d.sub.4) .delta. 8.55 (d, 1H, J=6.6 Hz), 7.95 (d, 1H, J=6.9
Hz), 7.63 (s, 1H), 7.20 (s, 1H), 3.93 (s, 3H), 3.78 (s, 3H), 2.35
(s, 3H), 2.30 (s, 3H), 2.17 (s, 3H), 1.63 (m, 1H), 0.97 (m, 1H),
0.85 (m, 3H). ESIMS m/z [M+H].sup.+ calculated=428.51.
found=428.58.
##STR00160##
4-(8-Methoxy-3-methyl-1-(2-methyl-1H-indol-3-yl)-5H-pyrido[4,3-b]indol-7--
yl)-3,5-dimethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4)
.delta. 7.69 (s, 1H), 7.58 (d, 1H, J=8.4 Hz), 7.52 (s, 1H),
7.26-7.32 (m, 1H), 7.11-7.19 (m, 2H), 6.69 (s, 1H), 3.23 (s, 3H),
2.88 (s, 3H), 2.59 (s, 3H), 2.31 (s, 3H), 2.12 (s, 3H). ESIMS m/z
[M+H].sup.+ calcd.=437.51. found=437.58.
##STR00161##
4-(8-Methoxy-3-methyl-1-(quinolin-4-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-d-
imethylisoxazole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 9.31
(d, 1H, J=4.2 Hz), 8.39 (d, 1H, J=8.4 Hz), 8.04 (d, 1H, J=4.5 Hz),
7.98-8.02 (m, 1H), 7.94 (d, 1H, J=0.6 Hz), 7.69 (d, 2H, J=3.6 Hz),
7.57 (s, 1H), 6.05 (s, 1H), 3.14 (s, 3H), 2.95 (d, 1H, J=0.3 Hz),
2.26 (s, 3H), 2.07 (s, 3H). ESIMS m/z [M+H].sup.+ calcd.=435.50.
found=435.67.
##STR00162##
4,4'-(8-Methoxy-3-methyl-5H-pyrido[4,3-b]indole-1,7-diyl)bis(3,5-dimethyl-
isoxazole). .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 7.81 (d, 1H,
J=0.6 Hz), 7.60 (s, 1H), 6.96 (s, 1H), 3.77 (s, 3H), 2.89 (s, 3H),
2.52 (s, 3H), 2.34 (s, 3H), 2.81 (s, 3H), 2.16 (s, 3H). ESIMS m/z
[M+H].sup.+ calcd.=403.45. found=403.67.
4. Synthesis of Final Compounds from the Other Intermediates
4.1 Synthesis of Demethoxylated Compounds:
##STR00163##
Synthetic methods are same to the reactions and conditions used in
the synthesis of RX3.
##STR00164##
4-(1-Chloro-5H-pyrido[4,3-b]indol-8-yl)-3,5-dimethylisoxazole.
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.40 (d, 1H, J=0.9 Hz),
8.31 (d, 1H, J=6.0 Hz), 7.77 (d, 1H, J=8.4 Hz), 7.62 (m, 2H), 2.49
(s, 1H), 2.33 (s, 1H). ESIMS m/z [M+H].sup.+ calcd.=298.75.
found=298.58.
##STR00165##
4,4'-(3-Methyl-5H-pyrido[4,3-b]indole-1,8-diyl)bis(3,5-dimethylisoxazole)-
. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 7.87-7.89 (M, 2H),
7.71 (dd, 1H, J.sub.1=8.4 Hz, J.sub.2=1.5 Hz), 7.28 (d, 1H, J=0.6
Hz), 2.90 (s, 3H), 2.47 (s, 3H), 2.41 (s, 3H), 2.24 (s, 3H), 2.22
(s, 3H). ESIMS m/z [M+H].sup.+ calcd.=373.43. found=373.67.
##STR00166##
4-(1-Chloro-3-methyl-5H-pyrido[4,3-b]indol-8-yl)-3,5-dimethylisoxazole.
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.34 (d, 1H, J=0.6 Hz),
7.75 (d, 1H, J=7.8 Hz), 7.60 (dd, 1H, J.sub.1=5.4 Hz, J.sub.2=1.8
Hz), 7.51 (s, 1H), 2.72 (s, 3H), 2.48 (s, 3H), 2.32 (s, 3H). ESIMS
m/z [M+H].sup.+ calcd.=312.77. found=313.17.
4.2 Synthesis of Final Compounds with 3,5-Dimethylisoxazole at a
Different Position.
##STR00167##
Synthetic methods are same to the reactions and conditions used in
the synthesis of RX3.
##STR00168##
6-bromo-1-chloro-8-methoxy-5H-pyrido[4,3-b]indole. .sup.1HNMR (300
MHz, MeOD-d.sub.4) .delta. 8.25 (d, J=6.0 Hz, 1H), 7.91 (d, J=2.4
Hz, 1H), 7.58 (d, J=6.0 Hz, 1H), 7.45 (d, J=2.1 Hz, 1H), 3.94 (s,
3H).
##STR00169##
4-(8-methoxy-5H-pyrido[4,3-b]indol-6-yl)-3,5-dimethylisoxazole.
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. MR (300 MHz, J=6.0 Hz),
8.04 (d, 1H, J=2.4 Hz), 7.49 (d, 1H, J=6.0 Hz), 7.16 (d, 1H, J=2.4
Hz), 3.98 (s, 3H), 2.36 (s, 3H), 2.20 (s, 3H). ESIMS m/z
[M+H].sup.+ calcd.=328.77. found=328.75.
4.3 Synthesis of Final Compounds Bearing Moieties in Addition to
3,5-dimethylisoxazole.
##STR00170##
Synthesis is same to the above-disclosed general Suzuki Coupling
method.
##STR00171##
1-chloro-8-methoxy-7-(1,3,5-trimethyl-1H-pyrazol-4-yl)-5H-pyrido[4,3-b]in-
dole. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.26 (d, 1H, J=6.0
Hz), 8.07 (s, 1H), 7.61 (d, 1H, J=6.3
Hz), 7.45 (s, 1H), 3.95 (s, 3H), 3.88 (s, 3H), 2.24 (s, 3H), 2.20
(s, 3H). ESIMS m/z [M+H].sup.+ calcd.=341.81. found=342.33.
##STR00172##
8-methoxy-1,7-bis(1,3,5-trimethyl-1H-pyrazol-4-yl)-5H-pyrido[4,3-b]indole-
. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.53 (d, 1H, J=6.6
Hz), 7.93 (d, 1H, J=6.6 Hz), 7.55 (s, 1H), 7.01 (s, 1H), 3.97 (s,
3H), 3.83 (s, 3H), 3.72 (s, 3H), 2.32 (s, 3H), 2.21 (s, 3H), 2.19
(s, 3H), 2.14 (s, 3H). ESIMS m/z [M+H].sup.+ calcd.=415.51.
found=415.58.
##STR00173##
1-chloro-7-(3,5-dimethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indol-
e. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.24 (d, 1H, J=6.0
Hz), 8.10 (s, 1H), 7.56 (d, 1H, J=6.0 Hz), 7.49 (s, 1H), 3.97 (s,
3H), 2.31 (s, 3H), 2.17 (s, 3H). ESIMS m/z [M+H].sup.+
calcd.=327.79. found=327.92.
##STR00174##
1,7-bis(3,5-dimethyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]indole.
.sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.55 (d, 1H, J=6.6 Hz),
7.96 (d, 1H, J=6.9 Hz), 7.66 (s, 1H), 7.03 (s, 1H), 6.32 (s, 1H),
3.73 (s, 3H), 2.40-2.29 (m, 12H). ESIMS m/z [M+H].sup.+ calcd.
=387.46. found=387.50.
##STR00175##
4-(1-chloro-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-5-ethyl-3-methylisoxazo-
le. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.19 (d, 1H, J=5.7
Hz), 8.08 (s, 1H), 7.49 (d, 1H, J=5.7
Hz), 7.43 (s, 1H), 3.96 (s, 3H), 2.64 (q, 2H, J=7.5 Hz), 2.34 (s,
3H), 1.12 (t, 3H, J=7.5 Hz). ESIMS m/z [M+H].sup.+ calcd.=342.80.
found=342.67.
##STR00176##
4-(1-chloro-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3-ethyl-5-methylisoxazo-
le. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.27 (d, 1H, J=6.3
Hz), 8.02 (s, 1H), 7.64 (d, 1H, J=6.3
Hz), 7.48 (s, 1H), 3.93 (s, 3H), 2.70 (q, 2H, J=7.5 Hz), 2.13 (s,
3H), 1.20 (t, 3H, J=7.5 Hz). ESIMS m/z [M+H].sup.+ calcd.=342.80.
found=342.42.
5. Synthesis of General Intermediate Containing
9H-pyrimido[4,5-b]indole Core
##STR00177##
S3 (2.26 g, 20 mmol) was dissolved in anhydrous DMF (50 mL) and the
solution was cooled to 0.degree. C. NaH (1.2 g, 60% in mineral oil,
30 mmol) was added in small portions. The resulting reaction
mixture was stirred for 0.5 h at 0.degree. C. and an anhydrous DMF
solution of known compounds S1 and S2 (20 mmol, ref. 2012, J. Med.
Chem. 55, 449-464) was added. The resulting solution was stirred at
0.degree. C. for 3 h before quenching with 1 N HCl. The aqueous
layer was extracted with ethyl acetate and combined organic layers
were washed with brine and dried over anhydrous Na.sub.2SO.sub.4.
The volatile components were removed on a rotary evaporator and the
residue was purified by flash column chromatogram. The desired
product S4 was isolated as colorless oil with impurity of the other
regioisomer (4.17 g, 64% yield). .sup.1HNMR (300 MHz, CDCl.sub.3):
8.41 (s, 1H), 7.11 (s, 1H), 5.60 (s, 1H), 4.24 (q, J=7.03 Hz, 2H),
4.01 (s, 3H), 1.25 (t, J=7.14 Hz, 3H).
##STR00178##
S4 (1.43 g, 4.2 mmol),
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(2.34 g, 10.5 mmol), and K.sub.2CO.sub.3 (2.03 g, 14.7 mmol) were
added to a round-bottom flask. DME (30 mL) and water (15 mL) were
added at room temperature. The solution was degassed, then
Pd(PPh.sub.3).sub.4 (242 mg, 0.21 mmol) was added in one portion.
The solution was again degassed, then heated at reflux for 14 h.
The aqueous layer was extracted with ethyl acetate, the combined
organic layers were washed with brine, then dried over anhydrous
Na.sub.2SO.sub.4. The volatile components were removed on a rotary
evaporator and the residue was purified by flash column
chromatogram. The desired product S5 was isolated in >80% yield
(1.47 g, contaminated with isomers and pinacol components). .sup.1H
NMR (CDCl.sub.3, 300 MHz): 8.10 (s, 1H), 7.27 (s, 1H), 5.78 (s,
1H), 4.35 (q, J=7.12 Hz, 2H), 3.99 (s, 3H), 2.33 (s, 3H), 2.18 (s,
3H), 1.37 (t, J=7.14 Hz, 3H).
##STR00179##
To an AcOH (30 mL) solution of S5 (1.47 g) at 80.degree. C., 0.8 g
Zn powder was added in small portions. The mixture was stirred at
80.degree. C. for 1 h, another 0.8 g Zn powder was added, and the
reaction was kept at the same temperature for 2 h. The reaction was
cooled, filtered, and washed with AcOH. The AcOH solution was
combined and the volatile components were removed on a rotary
evaporator. Purification by flash column chromatogram furnished the
desired product S6 (0.55 g, ca, 40% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 8.01 (br, s, 1H), 7.44 (s, 1H), 6.78 (s,
1H), 5.73 (br, s, 2H), 4.40 (q, J=7.08 Hz, 2H), 3.82 (s, 3H), 2.29
(s, 3H), 2.15 (s, 3H), 1.45 (t, J=7.08 Hz, 3H). ESI-MS calculated
for C.sub.17H.sub.20N.sub.3O.sub.4 [M+H].sup.+: 330.15, Obtained.
330.25.
##STR00180##
S6 (0.45 g, 1.4 mmol), ammonium formate (1.06 g, 17 mmol), and
formamide (16 mL) were heated at 175.degree. C. for 12 h. The
reaction was cooled to room temperature and water was added.
Filtration of the mixture yielded S7 as a brown solid (0.24 g, 0.77
mmol, 55% yield). .sup.1H NMR (DMSO-d6, 300 MHz): 8.09 (s, 1H),
7.57 (s, 1H), 7.24 (s, 1H), 3.81 (s, 3H), 3.30 (s, 1H), 2.62 (s,
3H), 2.06 (s, 3H), ESI-MS calculated for
C.sub.16H.sub.15N.sub.4O.sub.3 [M+H].sup.+: 311.11, Obtained:
311.75.
##STR00181##
S7 (0.24 g, 0.77 mmol) was dissolved in POCl.sub.3 (10 mL) and the
mixture was heated at 90.degree. C. for 5 h. The mixture was cooled
to room temperature and the volatile components were removed on a
rotary evaporator. Ethyl acetate (20 mL) was added at 0.degree. C.,
followed by NaHCO.sub.3 (20 mL) and water (20 mL). The mixture was
filtered and the desired CD54 product was collected as a brown
solid (0.17 g). The aqueous layer was extracted with ethyl acetate
and the combined organic layers were washed with brine and dried
over anhydrous Na.sub.2SO.sub.4. The volatile components were
removed on a rotary evaporator affording a brown solid (80 mg, 90
purity of CD54). .sup.1H NMR (DMSO-d6, 300 MHz): 8.74 (s, 1H), 7.84
(s, 1H), 7.45 (s, 1H). 3.89 (s, 3H), 3.31 (br, s, 1H), 2.29 (s,
3H), 2.09 (s, 3H) .sup.13C NMR (DMSO-d6, 75 MHz): 167.84, 161.17.
155.84, 122.24, 120.26, 116.96, 115.15, 113.11, 105.80, 57.84,
13.36, 12.39 ESI-MS calculated for
C.sub.16H.sub.14.sup.35ClN.sub.4O.sub.2 [M+H].sup.+: 329.08,
Obtained: 329.67.
Alternatively, S5 was also synthesized through a route showing
below:
##STR00182##
A mixture of known compounds S1 and S2 (ref. 2012, J. Med. Chem.
55, 449-464) (3.0 g, 12 mmol),
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(5.35 g, 24 mmol), and K.sub.2CO.sub.3 (5.0 g, 36 mmol) were added
to a round-bottom flask. DME (50 mL) and water (30 mL) were added
at room temperature. The solution was degassed before
Pd(PPh.sub.3).sub.4 (700 mg, 0.6 mmol) was added in one portion.
The solution was again degassed and then was heated at reflux for
14 h. The aqueous layer was extracted with ethyl acetate and
combined organic layers were washed with brine and dried over
anhydrous Na.sub.2SO.sub.4. The volatile components were removed on
a rotary evaporator and the residue was purified by flash column
chromatogram. The desired product S8 and S9 were isolated as a
mixture in >80% yield (3.38 g). The major isomer is compound S9,
.sup.1H NMR (CDCl.sub.3, 300 MHz): 8.03 (d, J=8.47 Hz, 1H), 6.93
(d, J=12.56 Hz, 1H), 4.00 (s, 3H), 2.40 (s, 3H), 2.24 (s, 3H).
S5 was synthesized by substitution of fluorine atom of S9 with
ethyl 2-cyanoacetate using NaH as a base and DMF as solvent. The
same reaction conditions to synthesize S5 from S4 was followed
(>80% isolated yield).
6. General Methods for Syntheses of Pinacol Boronates
The syntheses of pinacol boronates using n-butyl lithium via a
transmetalation intermediate is reported in the literature. The
procedures reported in following publications were adopted:
Synthesis, 2005, 20, 3581-3588, Synlett, 2006, 12, 1948-1952, and
J. Am. Chem. Soc, 2011, 133, 15800-15802.
##STR00183##
Four examples are illustrated below:
##STR00184##
Synthesis of tert-butyl
6-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate (R.dbd.H). tert-Butyl
6-fluoro-3-iodo-1H-indole-1-carboxylate (541 mg, 1.5 mmol) was
dissolved in anhydrous THF at -78.degree. C. BuLi (2.5 M THF
solution, 1.0 mL, 2.55 mmol) was added via a syringe and the
reaction was stirred for 20 min.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (558 mg, 3.0
mmol) was added via a syringe at -78.degree. C. and the reaction
was stirred for 2 h before quenching with saturated aqueous
NH.sub.4Cl solution. The aqueous layer was extracted with ethyl
acetate and the combined organic layers were washed with brine and
dried over anhydrous Na.sub.2SO.sub.4. The volatile components were
removed on a rotary evaporator the residue was purified by flash
column chromatography (0.35 g, 67% yield). .sup.1H NMR (CDCl.sub.3,
300 MHz): 8.09 (dd, J=8.96, 4.66 Hz, 1H), 8.02 (s, 1H), 7.64 (dd,
J=9.20, 2.57 Hz, 1H), 7.02 (dt, J=9.10, 2.60 Hz, 1H), 1.65 (s, 9H),
1.37 (s, 12H)
tert-Butyl
6-fluoro-2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indo-
le-1-carbo-xylate (R=Me, 82% yield) .sup.1H NMR (CDCl.sub.3, 300
MHz): 7.87 (dd, J=8.60, 5.84 Hz, 1H), 7.77 (dd, J=10.98, 2.41 Hz,
1H), 6.95 (dt, J=9.11, 2.42 Hz, 1H), 2.80 (s, 3H), 1.68 (s, 9H),
1.36 (s, 12H).
##STR00185##
tert-butyl
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo
[2,3-c]pyridine-1-carboxylate (R.dbd.H, 57% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 9.31 (s, 1H), 8.33 (d, J=5.34 Hz, 1H), 8.06
(s, 1H), 7.82 (dd, J=5.34, 0.97 Hz, 1H), 1.61 (s, 9H), 1.29 (s,
12H)
tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-c-
]pyridine-1-carboxylate (R=Me, 89% yield). .sup.1H NMR (CDCl.sub.3,
300 MHz): 9.25 (d, J=0.99 Hz, 1H), 8.36 (d, J=5.26 Hz, 1H), 7.86
(dd, J=5.26, 0.99 Hz, 1H), 2.88 (s, 3H), 1.71 (s, 9H), 1.38 (s,
12H).
In some cases, boronic acid and/or its pinacol esters were
synthesized through a transmetalation reaction promoted by i-PrMgCl
and LiCl complex (Boymond, L. et. al. Angew. Chem. Int. Ed. 1998,
37, No. 12, 1701-1703 and Hawkins, V. et. al. Organic Process
Research & Development 2008, 12, 1265-1268) following by adding
the Grignard reagents into isopropyl pinacol borate or triisopropyl
borate. For example, boronic acid pinacol ester CD164 was obtained
through a synthetic route showing below. Carboxylic acid CD157 was
synthesized following a previously reported method (Banno, H. et.
al. WO 2010/090716 A1). Acid CD157 was converted into CD164 in two
steps reaction following a previously reported method (Bethel, P.
A. et. al. 2012, Tetrahedron, 68, 5434-5444).
##STR00186##
CD157, .sup.1H NMR (DMSO-d6, 300 MHz): 12.32 (br, CO.sub.2H), 8.70
(d, J=6.91 Hz, 1H), 7.98 (d, J=8.85 Hz, 1H), 7.54-7.44 (m, 1H),
7.06-6.98 (m, 1H), 2.54 (s, 3H).
CD164, .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.39 (d, J=6.92 Hz, 1H),
8.84 (d, J=7.87 Hz, 1H), 7.15 (ddd, J=8.83, 6.77, 1.13 Hz, 1H),
6.72 (td, J=6.84, 1.39 Hz, 1H), 2.60 (s, 3H), 1.34 (s, 12H).
In some embodiments, the pinacol boronates prepared using this
method were not sufficiently stable for flash column
chromatography, and were used directly for next coupling step
without further purification.
The syntheses of pinacol boronates can also been achieved via
direct coupling of aryl halide and bis(pinacolato)diboron. The
procedures reported in following literatures were adopted: J. Org.
Chem. 1995, 7508-7510 and Angew. Chem. Int. Ed. 2007, 46,
5359-5363.
##STR00187##
Three examples are illustrated as below:
##STR00188##
tert-Butyl 3-iodo-2-methyl-1H-indole-1-carboxylate (1.0 g 4.0 mmol)
and bis(pinacolato)diboron were dissolved in dioxane. Et.sub.3N was
added via a syringe followed by Pd(dppf)Cl.sub.2. The reaction
mixture was refluxed for 3 h. The volatile components were removed
on a rotary evaporator the residue was dissolved in ether. The
mixture was filtered and ether solution was collected. The volatile
components were removed on a rotary evaporator, and the residue was
purified by flash column chromatography. tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate (>50% yield) was isolated with tert-butyl
2-methyl-1H-indole-1-carboxylate as impurity. Using the BuLi
method, the desired product was isolated in 67% yield (>90%
purity). .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.20-8.13 (m, 1H),
8.13-8.07 (m, 1H), 8.35-7.28 (m, 2H), 2.97 (s, 3H), 1.76 (s, 9H),
1.44 (s, 12H).
##STR00189##
The synthesis method for tert-butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate is same as that for tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate. The yield was >50% yield. .sup.1H NMR (CDCl.sub.3, 300
MHz): 7.90-7.80 (m, 1H), 7.45-7.35 (m, 1H), 7.25-7.10 (m, 2H), 2.66
(s, 3H), 1.38 (s, 9H).
##STR00190##
The synthesis method for CD143 is same as that for tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate. The isolated yield is 95% yield. .sup.1H NMR (CDCl.sub.3,
300 MHz): 8.49 (d, J=8.49 Hz, 1H), 7.50-7.38 (m, 2H), 7.28-7.18 (m,
1H), 4.17 (s, 3H), 1.25 (s, 12H).
7. Synthesis of Compounds from CD54
All final products were purified by reverse phase HPLC and the
products were in the form of CF.sub.3CO.sub.2H salt
(trifluoroacetic acid salt or TFA salt). In most cases, the counter
anion was not shown in the showing structures, unless otherwise
stated.
Some final products were synthesized via a Suzuki coupling as shown
in scheme below. Suzuki coupling used CD54 as the aryl halide
substrate, and commercially available or in-house made boronic
acids or pinacol boronates used as the coupling partners. The
reaction yields varied from 70% to 10%. Some pinacol boronates were
also synthesized using general methods shown in previous schemes.
One example of the Suzuki coupling procedure is illustrated in the
synthesis of Cpd. No. 35.
##STR00191##
Method A: CD54 (33 mg, 0.1 mmol),
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(66 mg, 0.3 mmol), and K.sub.2CO.sub.3 (70 mg, 0.5 mmol) were added
to a round-bottom flask. DME (6 mL) and water (4 mL) were added at
room temperature. The solution was degassed, then
Pd(PPh.sub.3).sub.4 (10-15 mg, 0.008-0.012 mmol) was added in one
portion. The solution was degassed again, then heated at reflux for
14 h. The aqueous layer was extracted with ethyl acetate and
combined organic layers were washed with brine and dried over
anhydrous Na.sub.2SO.sub.4. The volatile components were removed on
a rotary evaporator and the residue was purified by reverse phase
HPLC. The desired product Cpd. No. 35 TFA salt was isolated as a
colorless solid (16 mg, 41%).
##STR00192##
.sup.1H NMR (MeOD-d4, 300 MHz): 9.15 (s, 1H), 7.58 (s, 1H), 7.08
(s, 1H), 3.79 (s, 3H), 2.51 (s, 3H), 2.32 (s, 3H), 2.31 (s, 3H),
2.15 (s, 3H). ESI-MS calculated for C.sub.21H.sub.20N.sub.5O.sub.3
[M+H].sup.+: 390.16, Obtained: 390.42
Various compounds of the invention were synthesized via a direct
condensation of CD54 and an amine, alcohol, or thiol as shown
below. The reaction yields varied from 60% to 5%. One example of
the direct condensation procedure is illustrated in the synthesis
of Cpd. No. 36.
##STR00193##
Method B: CD54 (80 mg, 0.3 mmol), (R)-1-(pyridin-2-yl)ethanamine
(122 mg, 1 mmol), and EtN(i-Pr).sub.2 (0.3 mL, 1.5 mmol) were added
to a round-bottomed flask. NMP (3 mL) was added at room
temperature. The solution was heated at 140.degree. C. for 14 h,
then the reaction mixture was quenched by water (1 mL). The mixture
was purified by reverse phase HPLC. The desired product Cpd. No. 36
TFA salt was isolated as a brown solid (16 mg, 20%).
##STR00194##
Cpd. No. 36: .sup.1H NMR (MeOD-d4, 300 MHz): 8.81 (d, J=5.58 Hz,
1H), 8.54 (s, 1H), 8.56-8.47 (m, 1H), 8.23-8.17 (m, 1H), 8.19 (s,
1H), 7.92 (t, J=6.39 Hz, 1H), 7.50 (s, 1H), 6.00 (q, J=7.11 Hz,
1H), 4.02 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H), 2.00 (d, J=7.11 Hz,
3H). ESI-MS calculated for C.sub.23H.sub.23N.sub.6O.sub.2
[M+H].sup.+: 415.19, Obtained: 415.92.
##STR00195##
Method A-Suzuki coupling: 42% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.20 (s, 1H), 7.65 (s, 1H), 7.16 (s, 1H), 3.97 (s, 3H), 3.81
(s, 3H), 2.40 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H), 2.17 (s, 3H).
ESI-MS calculated for C.sub.22H.sub.23N.sub.6O.sub.2 [M+H].sup.+:
403.19, Obtained: 403.50.
##STR00196##
Method A--Suzuki coupling: 67% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 9.17 (s, 1H), 7.62 (s, 1H), 7.12 (s, 1H), 3.77 (s, 3H), 2.34
(s, 6H), 2.32 (s, 3H), 2.15 (s, 3H), ESI-MS calculated for
C.sub.21H.sub.21N.sub.6O.sub.2 [M+H].sup.+: 389.17, Obtained:
389.83.
##STR00197##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 37% yield. .sup.1H NMR (MeOD-d4, 300 MHz):
9.40 (s, 1H), 9.16 (s, 1H), 9.09 (s, 1H), 8.46 (d, J=6.54 Hz, 1H),
8.25 (d, J=6.54 Hz, 1H), 7.58 (s, 1H), 7.19 (s, 1H), 3.58 (s, 3H),
2.32 (s, 3H), 2.14 (s, 3H), ESI-MS calculated for
C.sub.23H.sub.10N.sub.6O.sub.2 [M+H].sup.+:411.16, Obtained:
411.42.
##STR00198##
Method B-Direct Condensation: 11% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 8.39 (s, 1H), 7.80 (s, 1H), 7.43 (s, 1H), 4.30-4.10 (m, 4H),
3.91 (s, 3H), 2.32 (s, 3H), 2.30-2.10 (m, 4H), 2.15 (s, 3H), ESI-MS
calculated for C.sub.20H.sub.22N.sub.5O.sub.2 [M+H].sup.+: 364.18,
Obtained: 364.46.
##STR00199##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 44% yield. .sup.1H NMR (MeOD-d4, 300 MHz):
9.10 (s, 1H), 8.38 (s, 1H), 7.80-7.70 (m, 1H), 7.62 (s, 1H),
7.30-7.10 (m, 3H), 3.52 (s, 3H), 2.35 (s, 3H), 2.16 (s, 3H), ESI-MS
calculated for C.sub.24H.sub.19FN.sub.5O.sub.2 [M+H].sup.+: 428.15,
Obtained: 428.25.
##STR00200##
Method A-Suzuki coupling: 7% yield. .sup.1H NMR (MeOD-d4, 300 MHz):
9.36 (s, 1H), 9.06 (s, 1H), 8.38 (s, 1H), 7.42 (s, 1H), 7.25 (s,
1H), 5.78 (s, 2H), 3.78 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H), ESI-MS
calculated for C.sub.26H.sub.23N.sub.6O.sub.2 [M+H].sup.+: 451.19,
Obtained: 451.25.
##STR00201##
Method B-Direct Condensation: 39% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 8.36 (s, 1H), 7.64 (s, 1H), 7.50-7.40 (m, 3H), 7.40-7.34 (m,
2H), 7.34-7.24 (m, 1H), 5.84 (t, J=6.13 Hz, 1H), 4.64-4.50 (m, 1H),
4.46-4.32 (m, 1H), 3.77 (s, 3H), 2.70-2.55 (m, 1H), 2.35-2.20 (m,
1H), 2.30 (s, 3H), 2.20-2.05 (m, 2H), 2.13 (s, 3H), ESI-MS
calculated for C.sub.26H.sub.26N.sub.5O.sub.2 [M+H].sup.+: 440.21,
Obtained: 440.50.
##STR00202##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 26% yield. .sup.1H NMR (MeOD-d4, 300 MHz):
9.12 (s, 1H), 7.61-7.58 (m, 1H), 7.58 (s, 1H), 7.37-7.15 (m, 3H),
6.82 (s, 1H), 3.32 (s, 3H), 2.67 (s, 3H), 2.31 (s, 3H), 2.12 (s,
3H), ESI-MS calculated for C.sub.25H.sub.22N.sub.5O.sub.2
[M+H].sup.+: 424.18, Obtained: 424.42.
##STR00203##
Method A-Suzuki coupling: 28% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 9.73 (s, 1H), 8.95 (s, 1H), 8.46 (s, 1H), 7.93 (s, 1H), 7.53
(s, 1H), 7.47 (s, 1H), 3.85 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H),
ESI-MS calculated for C.sub.19H.sub.17N.sub.6O.sub.2 [M+H].sup.+:
361.14, Obtained: 361.33.
##STR00204##
Method A-Suzuki coupling: 39% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 9.10 (s, 1H), 8.61 (dd, J=1.52, 0.88 Hz, 1H), 8.04 (t, J=1.52
Hz, 1H), 7.78 (s, 1H), 7.59 (s, 1H), 7.28 (dd, J=1.91, 0.88 Hz,
1H), 3.86 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H), ESI-MS calculated
for C.sub.20H.sub.17N.sub.4O.sub.3 [M+H].sup.+: 361.13, Obtained:
361.33.
##STR00205##
Method A-Suzuki coupling: 21% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 8.95 (s, 1H), 8.08 (s, 1H), 7.93 (t, J=1.65 Hz, 1H), 7.57 (s,
1H), 7.23 (dd, J=2.91, 1.81 Hz, 1H), 7.03 (dd, J=2.91, 1.62 Hz,
1H), 3.87 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H), ESI-MS calculated
for C.sub.20H.sub.18N.sub.5O.sub.2 [M+H].sup.+: 360.15, Obtained:
360.25.
##STR00206##
Method B-Direct Condensation: 42% yield. CD54, piperazine, and
(iPr).sub.2NEt were heated up at 180.degree. C. in DMF for 12 h.
HPLC purification yield Cpd. No. 48 as TFA salt. .sup.1H NMR
(MeOD-d4, 300 MHz): 8.55 (s, 1H), 8.19 (s, 1H), 7.46 (s, 1H), 7.41
(s, 1H), 4.10-3.90 (m, 4H), 3.97 (s, 3H), 3.90-3.80 (m, 2H),
3.80-3.70 (m, 2H), 2.33 (s, 3H), 2.16 (s, 3H). ESI-MS calculated
for C.sub.21H.sub.23N.sub.6O.sub.3 [M+H].sup.+: 407.18, Obtained:
407.33.
##STR00207##
Method B-Direct Condensation: 45% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 8.56 (s, 1H), 7.49 (s, 1H), 7.39 (s, 1H), 4.15-4.05 (m, 4H),
4.00-3.90 (m, 4H), 3.95 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS
calculated for C.sub.20H.sub.22N.sub.5O.sub.3[M+H].sup.+: 380.17,
Obtained: 380.50.
##STR00208##
Method B-Direct Condensation: 42% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 8.60 (s, 1H), 7.45 (s, 1H), 7.39 (s, 1H), 4.25-4.10 (m, 4H),
3.96 (s, 3H), 3.60-3.40 (m, 4H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS
calculated for C.sub.20H.sub.23N.sub.6O.sub.2 [M+H].sup.+: 379.19,
Obtained: 379.67.
##STR00209##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 40% yield. .sup.1H NMR (MeOD-d4, 300 MHz):
9.06 (s, 1H), 8.27 (s, 1H), 7.71 (d, J=8.20 Hz, 1H), 7.57 (s, 1H),
7.50 (d, J=7.96 Hz, 1H), 7.41 (ddd, J=8.24, 7.21, 1.13 Hz, 1H),
7.29 (ddd, J=8.03, 7.08, 0.97 Hz, 1H), 7.15 (s, 1H), 3.42 (s, 1H),
2.32 (s, 1H), 2.13 (s, 1H), ESI-MS calculated for
C.sub.24H.sub.20N.sub.5O.sub.2 [M+H].sup.+: 410.16, Obtained:
410.33.
##STR00210##
Method A-Suzuki coupling: 40% yield. .sup.1H NMR (MeOD-d4, 300
MHz): 9.05 (s, 1H), 8.89 (d, J=7.00 Hz, 1H), 8.76 (s, 1H), 7.76 (d,
J=7.85 Hz, 1H), 7.66-7.58 (m, 1H), 7.56 (s, 1H), 7.25 (dd, J=6.89,
6.92 Hz, 1H), 7.19 (s, 1H), 3.60 (s, 3H), 2.32 (s, 3H), 2.14 (s,
3H) ESI-MS calculated for C.sub.24H.sub.20N.sub.5O.sub.2
[M+H].sup.+: 410.16, Obtained: 410.12.
##STR00211##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 52% yield; .sup.1H NMR (MeOD-d4, 300 MHz):
12.04 (NH), 9.16 (s, 1H), 7.62 (s, 1H), 7.34 (dd, J=9.33, 2.16 Hz,
1H), 7.27 (dd, J=8.74, 5.06 Hz, 1H), 7.01 (ddd, J=3.42 (s, 3H),
2.67 (s, 3H), 2.33 (s, 3H), 2.14 (s, 3H).
##STR00212##
Method A-Suzuki coupling: 45% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.22 (s, 1H), 7.64 (s, 1H), 7.15 (s, 1H), 4.07 (s, 3H), 3.79
(s, 3H), 2.34 (s, 3H), 2.24 (s, 3H), 2.18 (s, 3H), 2.15-2.00 (m,
1H), 1.00-0.80 (m, 2H), 0.55-0.45 (m, 1H), 0.30-0.20 (m, 1H).
##STR00213##
Method A-Suzuki coupling: 22% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.20 (s, 1H), 7.63 (s, 1H), 7.37 (s, 1H), 3.92 (s, 3H), 3.84
(s, 3H), 2.39 (s, 3H), 2.35 (s, 3H), 2.17 (s, 3H), 1.80-1.65 (m,
1H), 1.25-1.15 (m, 1H), 1.00-0.80 (m, 3H).
##STR00214##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 61% yield; .sup.1H NMR (MeOD-d4, 300 MHz):
9.28 (s, 1H), 9.24 (s, 1H), 8.37 (d, J=6.53 Hz, 1H), 7.87 (d,
J=6.53 Hz, 1H), 7.63 (s, 1H), 6.77 (s, 1H), 3.47 (s, 3H), 2.85 (s,
3H), 2.32 (s, 3H), 2.14 (s, 3H).
##STR00215##
Method A-Suzuki coupling: 32% yield; Mixture of 2 isomers, ratio
1:1; .sup.1H NMR (MeOD-d.sub.4, 300 MHz): 9.17 (s, 1H), 9.06 (s,
1H), 7.56 (s, 1H), 7.51 (s, 1H), 7.50-7.40 (m, 1H), 7.40-7.30 (m,
6H), 7.25-7.15 (m, 3H), 7.15 (s, 1H), 7.00 (s, 1H), 4.06 (s, 3H),
4.00 (s, 3H), 3.82 (s, 3H), 3.68 (s, 3H), 2.41 (s, 3H), 2.32 (s,
3H), 2.31 (s, 3H), 2.26 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H).
##STR00216##
Method A-Suzuki coupling followed by treatment of trifluoroacetic
acid (2 mL) for 15 min: 65% yield; .sup.1H NMR (MeOD-d4, 300 MHz):
9.13 (s, 1H), 7.58 (s, 1H), 7.30 (s, 1H), 3.83 (s, 3H), 2.38 (s,
3H), 2.37 (s, 3H), 2.19 (s, 3H), 1.90-1.70 (m, 1H), 1.10-1.00 (m,
1H), 1.00-0.80 (m, 3H).
##STR00217##
Method B-Direct condensation: 45% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 8.49 (s, 1H), 7.65 (s, 1H), 7.49 (s, 1H), 3.96 (s, 3H), 3.64
(s, 6H), 2.34 (s, 3H), 2.18 (s, 3H).
##STR00218##
Method A-Suzuki coupling: 48% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.17 (s, 1H), 8.37 (d, J=8.58 Hz, 1H), 8.27 (s, 1H), 8.17 (t,
J=7.31 Hz, 1H), 8.03 (d, J=8.44 Hz, 1H), 7.82 (t, J=7.72 Hz, 1H),
7.49 (s, 1H), 6.43 (s, 1H), 3.30 (s, 3H), 3.12 (s, 3H), 2.27 (s,
3H), 2.08 (s, 3H).
##STR00219##
Method A-Suzuki coupling: 31% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.22 (s, 1H), 7.73 (d, J=8.32 Hz, 1H), 7.60 (s, 1H),
7.55-7.45 (m, 1H), 7.40-7.30 (m, 2H), 6.85 (s, 1H), 3.37 (s, 3H),
2.68 (s, 3H), 2.30 (s, 3H), 2.12 (s, 3H).
##STR00220##
Method A-Suzuki coupling: 26% yield; .sup.1H NMR (MeOD-d4, 300
MHz): 9.08 (s, 1H), 9.00 (d, J=6.10 Hz, 1H), 8.54 (s, 1H), 8.46
(dd, J=6.10, 1.36 Hz, 1H), 7.51 (s, 1H), 7.45 (s, 1H), 3.79 (s,
3H), 2.96 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H).
##STR00221##
Method B-Direct condensation of CD54 and benzimidazole in anhydrous
DMSO (4 mL) using EtN(i-Pr).sub.2 (0.1 mL) as base at 170.degree.
C. for 16 h. HPLC Isolated as TFA salt in 32% yield. .sup.1H NMR
(MeOD-d4, 300 MHz): 9.45 (s, 1H), 9.01 (s, 1H), 8.00 (d, J=8.09 Hz,
1H), 7.67-7.51 (m, 3H), 7.51 (s, 1H), 6.78 (s, 1H), 3.44 (s, 3H),
2.31 (s, 3H), 2.12 (s, 3H). ESI-MS Calculated for
C.sub.23H.sub.19N.sub.6O.sub.2 [M+H].sup.+=411.16. Found:
411.75.
##STR00222##
Optimized Suzuki coupling conditions previously reported (Jimenez,
J.-M. et. al. 2013, J. Med. Chem. DIO: 10.1021/jm301465a) was
followed to synthesize Cpd. No. 65. CD54 (34 mg, 0.1 mmol),
1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole
(CD143, 75 mg, 0.3 mmol), and Na.sub.2CO.sub.3 (50 mg) were mixed
in round-bottom flask. To this flask, MeOH (4 mL), PhMe (4 mL), and
water (1 mL) were added and the system was degassed and refilled
with pure nitrogen. Pd(PPh.sub.3).sub.4 (20 mg) was then added.
Again, the system was degassed and refilled with pure nitrogen. The
mixture was heated at reflux for 12 h. The aqueous layer was
extracted with ethyl acetate and the combined organic layers were
washed with brine and dried over anhydrous Na.sub.2SO.sub.4. The
volatile components were removed on a rotary evaporator and the
residue was purified by reverse phase HPLC. The desired product
Cpd. No. 65 was isolated as TFA salt in 23% yield. .sup.1H NMR
(DMSO-d6, 300 MHz): 12.36 (s, 1H), 9.31 (s, 1H), 9.05 (s, 1H), 8.82
(d, J=8.18 Hz, 1H), 7.85 (d, J=8.48 Hz, 1H), 7.60-7.50 (m, 1H),
7.39 (s, 1H), 7.40-7.32 (m, 1H), 4.36 (s, 3H), 4.00 (s, 3H), 3.37
(s, NH), 2.32 (s, 3H), 2.13 (s, 3H). ESI-MS Calculated for
C.sub.24H.sub.21N.sub.6O.sub.2 [M+H].sup.+=425.17. Found:
425.83.
##STR00223##
Coupling reaction of CD54 and benzotriazole catalyzed by
Pd.sub.2(dba).sub.3 [tris(dibenzylideneacetone)dipalladium(0)] and
[(.+-.)-BINAP
R.+-.)-1,1'-Binaphthalene-2,2'-diyl)bis(diphenylphosphine)] yielded
Cpd. No. 66. A previously reported methods by Ueda, S. et. al
(2012, J. Org. Chem. 77, 2543-2547) was adopted in this reaction
with following modification: BINAP was used as phosphine ligand
instead of the reported tBuXPhos (L3) or Me.sub.4tBuXPhos (L1).
CD54 (34 mg, 0.1 mmol), Benzotriazole (14 mg, 0.12 mmol), and
K.sub.3PO.sub.4 (42 mg, 0.2 mmol) were added into a round-bottom
flask. The round-bottom flask was degassed and refilled with pure
nitrogen gas. In a second round-bottom flask, Pd.sub.2(dba).sub.3
(9 mg, 0.01 mmol) and (.+-.)-BINAP (15 mg, 0.024 mmol) were added.
The round-bottom flask was degassed and refilled with pure nitrogen
gas. To this flask, anhydrous toluene (10 mL) was added and the
solution was heated at 120.degree. C. for 3 min to generate the
active catalyst. The active catalyst was transferred into the first
flask and the reaction mixture was heat at reflux for 12 h. The
mixture was then diluted with ethyl acetate and washed with water,
brine. Organic lawyer was collected, the volatile components were
removed on a rotary evaporator, and the residue was purified by
reverse phase HPLC. The desired product Cpd. No. 64 was isolated as
a TFA salt in 29% yield. .sup.1H NMR (DMSO-d6, 300 MHz): 12.88 (s,
1H), 9.04 (s, 1H), 8.61 (s, 1H), 8.26-8.18 (m, 2H), 7.68-7.60 (m,
2H), 7.48 (s, 1H), 3.87 (s, 3H), 3.30 (s, 3H), 3.15 (s, NH), 2.31
(s, 3H), 2.11 (s, 3H). ESI-MS Calculated for
C.sub.22H.sub.18N.sub.7O.sub.2 [M+H].sup.+=412.15. Found:
412.42.
8. Synthesis of General Intermediate Containing
9H-pyrido[3,4-b]indole Core and Compound Cpd. No. 67
##STR00224## ##STR00225##
A known acid T1 (1995, Tetrahedron, 51, 9531-9542.) was used as the
substrate to synthesize T2. The acid T1 (193 mg, 0.5 mmol) was
dissolved in AcOH--H.sub.2O (6 mL-4 mL) at room temperature.
PyHBr.sub.3 (160 mg, 0.5 mmol) was added in one portion. The
reaction was heated at 60.degree. C. for 12 h, then another portion
of PyHBr.sub.3 (80 mg, 0.25 mmol) was added. The reaction was
stirred at room temperature for 12 h. The reaction was quenched
with 0.1 mL sat. Na.sub.2SO.sub.3 and the volatile components were
removed on a rotary evaporator. MeOH was added, precipitate was
removed by filtration, and the MeOH solution was collected. The
volatile components were removed on a rotary evaporator and the
residue was purified by reverse phase HPLC. The desired product T2
was obtained in 36 mg, 15% yield. Side products T3 and T4 were
obtained in ca. 40% determined by UPLC based on conversion of
starting materials.
##STR00226##
.sup.1H NMR (CDCl.sub.3, 300 MHz): 9.29 (s, 1H), 9.08 (s, 1H), 7.84
(s, 1H), 6.62 (s, 1H), 3.87 (s, 3H). ESI-MS calculated for
C.sub.13H.sub.9.sup.79Br.sub.3NO.sub.3 [M+H].sup.+: 465.81,
Obtained: 465.84.
##STR00227##
The acid T2 (113 mg, 0.24 mmol), Et.sub.3N (excess, 0.5 mL), and
t-BuOH (10 mL) were added in a round-bottom flask at room
temperature. (PhO).sub.2PO--N.sub.3 (DPPA, 124 mg, 0.45 mmol) was
added in one portion, and the reaction was stirred at room
temperature for 2 h, then heated at reflux for 30 h. The volatile
components were removed on a rotary evaporator the residue was
purified by flash column chromatography. The desired product T5 was
isolated in 72 mg, 56% yield. .sup.1H NMR (CDCl.sub.3, 300 MHz):
9.28 (s, 1H), 8.53 (s, 1H), 7.91 (s, 1H), 6.68 (s, 1H), 5.95 (s,
1H), 3.88 (s, 3H), 1.46 (s, 9H), ESI-MS calculated for
C.sub.17H.sub.18.sup.81Br.sub.2.sup.79BrN.sub.2O.sub.3 [M+H].sup.+:
538.88, Obtained: 538.92.
##STR00228##
The substrate T5 (72 mg, 0.2 mmol), CuI (76 mg, 0.3 mmol), and NaH
(40 mg, 0.4 mmol, 60% in mineral oil) were placed in an oven-dried
round-bottom flask Anhydrous diglyme (10 ml) was added via a
syringe and the reaction mixture was heated at 120.degree. C. for
14 h. The reaction mixture was quenched with 5% NH.sub.3/H.sub.2O.
The aqueous layer was extracted with ethyl acetate, and combined
organic layers were washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. The volatile components were removed on a rotary
evaporator and the residue was purified by flash column
chromatography. The desired product T6 was isolated in 22 mg, 52%
yield. .sup.1H NMR (DMSO-d6, 300 MHz): 11.92 (NH, 1H), 8.89 (s,
1H), 8.41 (s, 1H), 8.10 (s, 1H), 7.94 (s, 1H), 3.94 (s, 3H).
.sup.13C NMR (DMSO-d.sub.6, 75 MHz): 155.70, 149.19, 138.98,
137.27, 135.70, 133.43, 119.53, 116.43, 113.92, 104.06, 94.54,
56.54. ESI-MS calculated for C.sub.12H.sub.9.sup.79Br.sub.2N.sub.2O
[M+H].sup.+:356.91, Obtained: 357.58.
##STR00229##
T6 (22 mg, 0.06 mmol),
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(44 mg, 0.2 mmol), and K.sub.2CO.sub.3 (55 mg, 0.4 mmol) were
placed in a round-bottom flask. DME (6 mL) and water (4 mL) were
added, and the solution was degassed then Pd(PPh.sub.3).sub.4 (10
mg, 0.008 mmol) was added in one portion. The solution was degassed
again, then heated at reflux for 14 h. The aqueous layer was
extracted with ethyl acetate and the combined organic layers were
washed with brine and dried over anhydrous Na.sub.2SO.sub.4. The
volatile components were removed on a rotary evaporator and the
residue was purified by reverse phase HPLC. The desired product
Cpd. No. 67 TFA salt was isolated as a yellow-green solid (27 mg,
TFA salt, >90% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 9.26 (s,
1H), 8.45 (s, 1H), 7.71 (s, 1H), 7.03 (s, 1H), 3.74 (s, 3H), 2.41
(s, 3H), 2.33 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H). ESI-MS
calculated for C.sub.22H.sub.21N.sub.4O.sub.3 [M+H].sup.+: 389.16,
Obtained: 389.42
9. Synthesis of General Intermediate Containing
5H-pyridazino[4,5-b]indole Core
##STR00230## ##STR00231##
10. Synthesis of General Intermediate Containing
2-Methyl-9H-pyrimido[4,5-b]indole Core
##STR00232##
To a round-bottom flask, S6 (0.37 g, 1.1 mmol) and MeCN (20 mL)
were added at room temperature. Dry HCl was bubbled through MeCN
for 30 min and the reaction mixture was warmed up to reflux (ca,
82.degree. C.) for 2.5 h. The reaction was then cooled to room
temperature and the volatile components were removed on a rotary
evaporator. To this crude mixture, 10% NaOH aqueous solution (20
mL) and EtOH (50 mL) were added and the solution was heated at
reflux for 6 h. The volatile components were then removed on a
rotary evaporator and the aqueous residue was acidified with 2N HCl
aqueous solution. The product S12 was allowed to precipitate at
0.degree. C. Filtration of the mixture furnished pure S12 in 0.278
g (78% yield, 2 steps). .sup.1H NMR (DMSO-d6, 300 MHz): 7.57 (s,
1H), 7.20 (s, 1H), 3.81 (s, 3H), 2.37 (s, 3H), 2.27 (s, 3H), 2.08
(s, 3H).
##STR00233##
To a round-bottom flask, S12 (0.278 g, 0.8 mmol) and POCl.sub.3 (8
mL) were added. The mixture was heated at 90.degree. C. for 6 h.
The reaction mixture was cooled to room temperature and the
volatile components were removed on a rotary evaporator. Water (20
mL) and ethyl acetate (20 mL) were added and the pH was adjusted to
8 using NaHCO.sub.3 saturated aqueous solution. Filtration of the
mixture furnished S13 as a brown solid in 0.208 g (75% yield).
.sup.1H NMR (DMSO-d6, 300 MHz): 7.81 (s, 1H), 7.43 (s, 1H), 3.89
(s, 3H), 2.69 (s, 3H), 2.31 (s, 3H), 2.11 (s, 3H).
Some final products were synthesized via a Suzuki coupling as shown
in the scheme below. Suzuki coupling used S13 as the aryl halide
substrate, and commercially available or in-house made boronic
acids or pinacol boronates used as the coupling partners. The
reaction yields varied from 70% to 10%. Some pinacol boronates were
also synthesized using general methods shown in previous schemes.
One example of the Suzuki coupling procedure is illustrated in the
synthesis of Cpd. No. 68.
##STR00234##
Method C:
S13 (34 mg, 0.1 mmol), tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate (100 mg, 0.3 mmol), and K.sub.2CO.sub.3 (70 mg, 0.4 mmol)
were added to a round-bottom flask. DME (6 mL) and water (4 mL)
were added at room temperature. The solution was degassed, then
Pd(PPh.sub.3).sub.4 (20 mg, 0.017 mmol) was added in one portion.
The solution was again degassed, following by heat-up at reflux for
14 h. The aqueous layer was extracted with ethyl acetate and
combined organic layers were washed with brine and dried over
anhydrous Na.sub.2SO.sub.4. The volatile components were removed on
a rotary evaporator and the residue was treated with
trifluoroacetic acid (2 mL) for 15 min at room temperature.
Purification by reverse phase HPLC afforded desired product Cpd.
No. 68 TFA salt was isolated as a colorless solid (12 mg, 27%).
##STR00235##
Cpd. No. 68: .sup.1H NMR (MeOD-d4, 300 MHz): 11.94 (NH), 7.62 (d,
J=8.13 Hz, 1H), 7.57 (s, 1H), 7.35 (ddd, J=8.17, 6.82, 1.30, 1H),
7.32-7.18 (m, 2H), 6.80 (s, 1H), 3.00 (s, 3H), 2.70 (s, 3H), 2.34
(s, 3H), 2.15 (s, 3H).
##STR00236##
General Suzuki coupling reaction condition (Method C) was followed:
37% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 8.95 (d, J=7.00 Hz, 1H),
8.76 (s, 1H), 7.70-7.64 (m, 2H), 7.56 (s, 1H), 7.34-7.24 (m, 1H),
6.98 (s, 1H), 3.54 (s, 3H), 2.96 (s, 3H), 2.31 (s, 3H), 2.13 (s,
3H). ESI-MS Calculated for C.sub.24H.sub.21N.sub.6O.sub.2
[M+H].sup.+=425.17. Found: 425.42.
##STR00237##
General Suzuki coupling reaction condition (Method C) was followed:
30% yield; .sup.1H NMR (MeOD-d.sub.4, 300 MHz): 11.96 (s, NH), 7.54
(s, 1H), 7.40-7.20 (m, 2H), 7.10-6.90 (m, 1H), 6.77 (s, 1H), 3.38
(s, 3H), 2.96 (s, 3H), 2.63 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H).
ESI-MS Calculated for C.sub.26H.sub.23FN.sub.5O.sub.2
[M+H].sup.+=456.18. Found: 456.33.
##STR00238##
General Suzuki coupling reaction condition (Method C) was followed:
47% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 9.28 (s, 1H), 8.39 (d,
J=6.53 Hz, 1H), 7.90 (d, J=6.50 Hz, 1H), 7.58 (s, 1H), 6.69 (s,
1H), 3.45 (s, 3H), 3.00 (s, 3H), 2.82 (s, 3H), 2.30 (s, 3H), 2.11
(s, 3H). ESI-MS Calculated for C.sub.25H.sub.23N.sub.6O.sub.2
[M+H].sup.+=439.19. Found: 439.58.
##STR00239##
General Suzuki coupling reaction condition (Method C) was followed:
40% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 11.37 (s, NH), 7.89 (d,
J=8.14 Hz, 1H), 7.64 (dd, J=7.34, 0.89 Hz, 1H), 7.56-7.43 (m, 2H),
7.52 (s, 1H), 6.73 (s, 1H), 6.31 (s, 1H), 3.37 (s, 3H), 2.98 (s,
3H), 2.29 (s, 3H), 2.10 (s, 3H). ESI-MS Calculated for
C.sub.25H.sub.22N.sub.5O.sub.2 [M+H].sup.+=424.18. Found:
424.42.
##STR00240##
General Suzuki coupling reaction condition (Method C) was followed:
57% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 9.31 (d, J=4.59 Hz, 1H),
8.38 (d, J=8.50 Hz, 1H), 8.08 (d, J=4.63 Hz, 1H), 8.12-8.00 (m,
1H), 7.88 (d, J=7.76 Hz, 1H), 7.78-7.70 (m, 1H), 7.53 (s, 1H), 6.21
(s, 1H), 3.21 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H).
ESI-MS Calculated for C.sub.26H.sub.22N.sub.5O.sub.2
[M+H].sup.+=436.18. Found: 436.50.
##STR00241##
General Suzuki coupling reaction condition (Method C) was followed:
23% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 7.81 (d, J=8.10 Hz, 1H),
7.59 (d, J=8.10 Hz, 1H), 7.55 (s, 1H), 7.50-7.30 (m, 1H), 7.38 (s,
1H), 7.25 (t, J=8.10 Hz, 1H), 3.66 (s, 3H), 2.97 (s, 3H), 2.53 (s,
3H), 2.31 (s, 3H), 2.14 (s, 3H). ESI-MS Calculated for
C.sub.26H.sub.24N.sub.5O.sub.2 [M+H].sup.+=438.19. Found:
438.67.
##STR00242##
General Suzuki coupling reaction condition (Method C) was followed:
22% yield; .sup.1H NMR (MeOD-d4, 300 MHz): 8.83 (s, J=6.97 Hz, 1H),
7.64-7.54 (m, 1H), 7.59 (s, 1H), 7.50 (d, J=8.82 Hz, 1H), 7.22 (td,
J=6.81, 1.46 Hz, 1H), 6.74 (s, 1H), 3.50 (s, 3H), 2.99 (s, 3H),
2.65 (s, 3H), 2.33 (s, 3H), 2.14 (s, 3H). ESI-MS Calculated for
C.sub.25H.sub.23N.sub.6O.sub.2 [M+H].sup.+=439.19. Found:
440.83.
S13 (30 mg), 3-quinoline boronic acid (60 mg), and K.sub.2CO.sub.3
(64 mg) were placed in a round-bottom flask. To this flask,
1,2-dimethoxyethane (DME, 6 mL) and water (4 mL) were added and the
system was degassed to remove oxygen. Pd(PPh.sub.3).sub.4 (20 mg)
was added in one portion and the flask was degassed again. The
mixture was heated at reflux for 12 h under nitrogen atmosphere.
The reaction was then diluted with water and the aqueous layer was
extracted with ethyl acetate (50 mL.times.2) and the combined
organic layers were dried over anhydrous sodium sulfate. The
solvent was removed in vacuum and the residue was purified by
reverse phase preparative HPLC to yield the desired product Cpd.
No. 76 in 22% yield as a salt of CF.sub.3CO.sub.2H.
The following compounds were prepared in following the same Suzuki
coupling method [(Pd(PPh.sub.3).sub.4 as catalyst and
K.sub.2CO.sub.3 as base]. The boronic acids required for these
syntheses are commercially available.
##STR00243##
TFA salt .sup.1H NMR (300 MHz, MeOD-d4): 9.47 (d, J=2.01 Hz, 1H),
9.21 (d, J=2.03 Hz, 1H), 8.29 (t, J=7.52 Hz, 2H), 8.08 (ddd,
J=8.50, 6.99, 1.40 Hz, 1H), 7.88 (ddd, J=8.05, 7.14, 1.00 Hz, 1H),
7.57 (s, 1H), 7.29 (s, 1H), 3.60 (s, 3H), 3.00 (s, 3H), 2.31 (s,
3H), 2.13 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.22N.sub.5O.sub.2 [M+H].sup.+=436.18, Obtained:
436.83.
##STR00244##
TFA salt yield: 27%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 9.15 (dd, J=4.39, 1.39 Hz, 1H), 8.61-8.53 (m,
1H), 8.41 (d, J=8.60 Hz, 1H), 8.25 (d, J=1.33 Hz, 1H), 8.24 (s,
1H), 7.72 (dd, J=7.74, 4.42 Hz, 1H), 7.57 (s, 1H), 6.30 (s, 1H),
3.28 (s, 3H), 3.03 (3H), 2.29 (s, 3H), 2.10 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.22N.sub.5O.sub.2 [M+H].sup.+=436.18,
Obtained: 436.33.
##STR00245##
TFA salt yield: 36%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 9.08 (d, J=4.56 Hz, 1H), 8.46 (J=8.81 Hz, 1H),
8.22 (d, J=8.56 Hz, 1H), 8.17 (d, J=8.86 Hz, 1H), 7.69 (dd, J=8.63,
4.64 Hz, 1H), 7.55 (s, 1H), 6.14 (s, 1H), 3.28 (s, 3H), 3.01 (s,
3H), 2.47 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated
for C.sub.27H.sub.24N.sub.5O.sub.2 [M+H].sup.+=450.19, Obtained:
450.50.
##STR00246##
TFA salt yield: 54%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 9.78 (s, 1H), 8.98 (s, 1H), 8.53-8.46 (m, 1H),
8.04-7.92 (m, 2H), 7.90-7.82 (m, 1H), 7.55 (s, 1H), 6.23 (s, 1H),
3.20 (s, 3H), 3.01 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.22N.sub.5O.sub.2 [M+H].sup.+=436.18,
Obtained: 436.50.
##STR00247##
TFA salt yield: 29%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 9.85 (s, broad, 1H), 8.80 (d, J=8.32 Hz, 1H),
8.75-8.50 (broad, 1H), 8.56 (dd, J=7.24, 1.11 Hz), 8.26 (dd,
J=8.29, 7.30 Hz, 1H), 8.04 (d, J=6.08 Hz, 1H), 6.32 (s, 1H), 3.29
(s, 3H), 3.00 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.22N.sub.5O.sub.2 [M+H].sup.+=436.18,
Obtained: 436.68.
##STR00248##
TFA salt yield: 35%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 9.54 (s, 1H), 8.74 (s, 1H), 8.61 (dd, J=7.75,
1.22 Hz, 1H), 8.52 (d, J=6.18 Hz, 1H), 8.47-8.35 (m, 2H), 7.54 (s,
1H), 6.42 (s, 1H), 3.32 (s, 3H), 2.99 (s, 3H), 2.26 (s, 3H), 2.08
(s, 3H). ESI-MS calculated for C.sub.26H.sub.22N.sub.5O.sub.2
[M+H].sup.+=436.18, Obtained: 436.56.
##STR00249##
TFA salt yield: 18%. Suzuki
coupling-Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 Method. .sup.1H NMR
(300 MHz, MeOD-d4): 8.89 (dd, J=4.28, 1.72 Hz, 1H), 8.65 (dd,
J=8.41, 1.68 Hz, 1H), 8.47 (dd, J=8.28, 1.31 Hz, 1H), 8.37 (dd,
J=7.18, 1.36 Hz, 1H), 8.02 (dd, J=8.20, 7.26 Hz, 1H), 7.74 (dd,
J=8.38, 4.28 Hz, 1H), 7.54 (s, 1H), 6.43 (s, 1H), 3.33 (s, 3H),
2.99 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.22N.sub.5O.sub.2 [M+H].sup.+=436.18, Obtained:
436.83.
##STR00250##
1H-Indole-2-carbonitrile (0.5 g) was dissolved in DMF (10 mL) at
room temperature. The solution was cooled with a water-ice bath.
N-Iodosuccinimide (NIS, 0.9 g) was added in small portions. The
reaction was stirred at room temperature for 12 h before quenching
with water. The aqueous layer was extracted with ethyl acetate (100
mL.times.3) and the combined organic layers were washed with water
(40 mL.times.5) and dried over anhydrous sodium sulfate. The
solvent was removed on a rotary evaporator. The residue was placed
in a round-bottom flask and Boc.sub.2O (3.1 g) and THF (20 mL) were
added. DMAP (900 mg) was then added in small portions. The reaction
was stirred at room temperature for 12 h. The solvent was removed
on a rotary evaporator and the residue was purified by flash column
chromatography to yield CD174 (1.52 g, 82% yield). .sup.1H NMR (300
MHz, CDCl.sub.3): 8.23 (d, J=8.52 Hz, 1H), 7.60-7.46 (m, 2H),
7.44-7.36 (m, 1H), 1.73 (s, 9H).
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.6 g) and
CD174 (0.7 g), and THF (10 m) were placed in a round-bottom flask.
The solution was cooled with a water-ice bath. iPrMgCl--LiCl
complex solution in THF (1.3 M, 1.60 mL) was added via a syringe.
The reaction was stirred at room temperature for 3 h. The reaction
mixture was quenched with saturated aqueous NH.sub.4Cl solution.
The aqueous layer was extracted with ethyl acetate (100 mL.times.3)
and the combined organic layers were washed with brine and dried
over anhydrous sodium sulfate. The solvent was removed on a rotary
evaporator and the residue was purified by flash column
chromatography to yield CD182 (0.18 g). .sup.1H NMR (300 MHz,
CDCl.sub.3): 8.19 (d, J=8.44 Hz, 1H), 8.05 (d, J=7.88 Hz, 1H), 7.47
(t, J=7.74 Hz, 1H), 7.34 (t, J=7.50 Hz, 1H), 1.73 (s, 9H), 1.41 (s,
12H).
Suzuki coupling of S13 and CD182 using
Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 method and subsequent
de-protection of Boc group in TFA-CH.sub.2Cl.sub.2 furnished Cpd.
No. 90 in 2% yield after HPLC purification as a salt of
CF.sub.3CO.sub.2H.
##STR00251##
.sup.1H NMR (300 MHz, MeOD-d4): 7.76 (dt, J=8.46, 0.79 Hz, 1H),
7.60 (ddd, J=8.40, 6.93, 1.11 Hz, 1H), 7.54 (dt, J=8.09, 0.95 Hz,
1H), 7.55 (s, 1H), 7.42-7.35 (m, 1H), 6.78 (s, 1H), 3.35 (s, 3H),
2.99 (s, 3H), 2.30 (s, 3H), 2.12 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.21N.sub.6O.sub.2 [M+H].sup.+=449.17, Obtained:
449.67
##STR00252##
Ph.sub.3P--Br.sub.2 (prepared from 2.4 g Br.sub.2 and 3.93 g
PPh.sub.3 in CH.sub.2Cl.sub.2, see reference J. Org. Chem. 1976, V
41, No. 20, p. 3279) was dissolved in MeCN.
2-Methyl-4-hydroxyquinoline (1.5 g) was added in one portion and
the mixture was heated at reflux for 2 h. Solvent was removed and
the residue was purified by flash column chromatography to furnish
4-bromo-2-methylquinoline CD229 in 0.60 g (41% yield). CD229 has
also been prepared by reflux toluene solution of
2-methyl-4-hydroxyquinoline and POBr.sub.3 for 4 h.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5.20 g),
4-bromo-2-methylquinoline (2.23 g), and THF (40 m) were placed in a
round-bottom flask. The solution was cooled with a dry ice-ethanol
bath at -78.degree. C. BuLi (2.5 M THF solution, 7.2 mL) was added
via a syringe. The reaction was stirred at -78.degree. C. for 3 h
before quenching with statured aqueous NH.sub.4Cl solution. The
aqueous layer was extracted with ethyl acetate (100 mL.times.3) and
the combined organic layers were washed with brine and dried over
anhydrous sodium sulfate. The solvent was removed on a rotary
evaporator and the residue was purified by flash column
chromatography to furnish 2-methylquinoline-4-boronic acid pinacol
ester CD231 in 1.67 g (62% yield). .sup.1H NMR (300 MHz,
CDCl.sub.3): 8.58 (d, J=8.31 Hz, 1H), 8.02 (d, J=8.43 Hz, 1H), 7.66
(t, J=7.63 Hz, 1H), 7.51 (t, J=7.60 Hz, 1H), 7.26 (s, 1H), 2.74 (s,
3H), 1.43 (s, 12H). ESI-MS calculated for C.sub.16H.sub.21BNO.sub.2
[M+H].sup.+=270.17, observed: 270.83.
S13 (728 mg), 2-methylquinoline-4-boronic acid pinacol ester (1.67
g), 1,2-dimethoxyethane (60 mL), and Na.sub.2CO.sub.3 (20 mL, 2 M
aqueous solution) were mixed in a round-bottom flask and the system
was degassed to remove oxygen. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2
complex (257 mg) was added on one portion and the system was
degassed again. The mixture was heated at reflux for 12 h under
nitrogen atmosphere. The reaction was then diluted with water and
the aqueous layer was extracted with ethyl acetate (100 mL.times.3)
and the combined organic layers were dried over anhydrous sodium
sulfate. The solvent was removed in vacuum and the residue was
purified by flash column chromatography to yield the desired
product Cpd. No. 84 in 0.64 g (63% yield). Further purification was
aided by a reverse phase HPLC to yield the corresponding products
as a salt of CF.sub.3CO.sub.2H.
##STR00253##
.sup.1H NMR (300 MHz, MeOD-d4): 8.32 (d, J=8.47 Hz, 1H), 8.13 (s,
1H), 8.10 (ddd, J=8.44, 7.03, 1.26 Hz, 1H), 7.93 (d, J=7.86 Hz,
1H), 7.75 (t, J=7.71 Hz, 1H), 7.49 (s, 1H), 6.30 (s, 1H), 3.25 (s,
3H), 3.04 (s, 3H), 2.95 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.27H.sub.24N.sub.5O.sub.2 [M+H].sup.+=450.19,
Obtained: 450.42.
The following compounds were prepared using the same Suzuki
coupling reaction conditions (sodium carbonate as base and
(Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex as catalyst) for the
preparation of Cpd. No. 84. Their purification was performed on a
reverse phase HPLC to yield the corresponding products as a salt of
CF.sub.3CO.sub.2H.
##STR00254##
2-Trifluoromethyl-4-bromoquinoline (500 mg) was dissolved in
anhydrous THF (15 mL). The solution was cooled to -78.degree. C. in
a dry ice-ethanol bath. BuLi (0.94 mL, 2.5 M in THF) was added
dropwise and the mixture was stirred at -78.degree. C. for 15 min.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (506 mg) was
added via a syringe and the reaction mixture was stirred at
-78.degree. C. for 3 h before quenching with statured aqueous
NH.sub.4Cl solution. The aqueous layer was extracted with ethyl
acetate (50 mL.times.3) and the combined organic layers were washed
with brine and dried over anhydrous sodium sulfate. The solvent was
removed on a rotary evaporator and the residue was purified by
flash column chromatography to furnish 2-trifluoromethylquinoline
4-boronic acid pinacol ester CD194 in 0.35 g (60% yield). .sup.1H
NMR (300 MHz, CDCl.sub.3): 8.74 (d, J=7.81 Hz, 1H), 8.22 (d, J=8.46
Hz, 1H), 7.80 (ddd, J=8.39, 6.85, 1.28 Hz, 1H), 7.69 (ddd, J=8.55,
7.04, 1.26 Hz, 1H), 1.45 (s, 12H).
Cpd. No. 85-TFA salt was prepared from Suzuki coupling of CD194 and
S13 using Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex
--Na.sub.2CO.sub.3 (2 M) condition. 10% yield
##STR00255##
.sup.1H NMR (300 MHz, MeOD-d4): 8.44 (d, J=8.61 Hz, 1H), 8.28 (s,
1H), 8.04 (t, J=7.71 Hz, 1H), 7.90 (d, J=8.19 Hz, 1H), 7.76 (t,
J=8.03 Hz, 1H), 7.45 (s, 1H), 6.20 (s, 1H), 3.21 (s, 3H), 2.93 (s,
3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for
C.sub.27H.sub.21F.sub.3N.sub.5O.sub.2 [M+H].sup.+=504.16, Obtained:
504.58.
##STR00256##
2-Trifluoromethyl-1H-indole (0.5 g) was dissolved in DMF (15 mL) at
room temperature. The solution was cooled with a water-ice bath.
N-Iodosuccinimide (NIS, 0.726 g) was added in small portions. The
reaction was stirred at room temperature for 12 h before quenching
with water. The aqueous layer was extracted with ethyl acetate (100
mL.times.3) and the combined organic layers were washed with water
(40 mL.times.5) and dried over anhydrous sodium sulfate. The
solvent was removed on a rotary evaporator. The residue was placed
in a round-bottom flask and Boc.sub.2O (1.18 g) and THF (20 mL)
were added. DMAP (488 mg) was then added in small portions. The
reaction was stirred at room temperature for 12 h. The solvent was
removed on a rotary evaporator and the residue was purified by
flash column chromatography to yield CC52 (1.52 g, 91% yield over
two steps). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.13 (d, J=7.94 Hz,
1H), 7.57 (d, J=7.94 Hz, 1H), 7.49 (t, J=7.49 Hz, 1H), 7.37 (t,
J=7.53 Hz, 1H), 1.65 (s, 9H).
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (690 g), CC52
(1.015 g), and THF (20 m) were placed in a round-bottom flask. The
solution was cooled with an ice-water bath. iPrMgCl--LiCl (1.3 M
THF solution, 2.50 mL) was added via a syringe. The reaction was
stirred at 0.degree. C. for 3 h. The reaction mixture was quenched
with statured aqueous NH.sub.4Cl solution. The aqueous layer was
extracted with ethyl acetate (50 mL.times.3) and the combined
organic layers were washed with brine and dried over anhydrous
sodium sulfate. The solvent was removed on a rotary evaporator and
the residue was purified by flash column chromatography to yield
CD176 in 0.67 g (66% yield). .sup.1H NMR (300 MHz, CDCl.sub.3):
8.18 (d, J=8.45 Hz, 1H), 7.77 (d, J=7.81 Hz, 1H), 7.42 (t, J=7.81
Hz, 1H), 7.29 (t, J=7.55 Hz, 1H), 1.66 (s, 9H), 1.42 (s, 12H).
Cpd. 86-TFA salt was prepared from Suzuki coupling of CD176 and S13
using Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3
(2 M) condition. 5% yield.
##STR00257##
.sup.1H NMR (300 MHz, MeOD-d4): 7.75 (d, J=8.28 Hz, 1H), 7.55-7.48
(m, 1H), 7.51 (s, 1H), 7.39 (d, J=8.06 Hz, 1H), 7.28 (t, J=8.07 Hz,
1H), 6.48 (s, 1H), 3.24 (s, 3H), 2.94 (s, 3H), 2.28 (s, 3H), 2.09
(s, 3H). ESI-MS calculated for
C.sub.26H.sub.21F.sub.3N.sub.5O.sub.2 [M+H].sup.+=492.16, Obtained:
492.42.
##STR00258##
3-Bromo-2-methylimidazo[1,2-a]pyridine (2.11 g) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.98 g) were
dissolved in anhydrous THF (40 mL) and the solution was cooled with
an ice-water bath. iPrMgCl--LiCl (1.3 M in THF, 10 mL) was added
via a syringe pump over 30 min. The reaction was stirred for 2.5 h
before quenching with saturated aqueous NH.sub.4Cl solution. The
aqueous layer was extracted with ethyl acetate and the combined
organic layers was washed with brine and dried over anhydrous
sodium sulfate. The residue was purified by flash column
chromatography to furnish the desired boronate CD168 in 1.25 g (48%
yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.81 (d, J=6.63 Hz, 1H),
7.54 (d, J=8.85 Hz, 1H), 7.25-7.16 (m, 1H), 6.78 (t, J=6.67 Hz,
1H), 2.63 (s, 3H), 1.37 (s, 12H).
Suzuki coupling of S13 and CD168 under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
furnished Cpd. No. 87-TFA salt in <7% yield.
##STR00259##
.sup.1H NMR (300 MHz, MeOD-d4): 8.75 (d, J=6.78 Hz, 1H), 8.08 (dd,
J=2.18, 1.05 Hz, 1H), 8.06 (d, J=1.04 Hz, 1H), 7.54-7.46 (m, 1H),
7.48 (s, 1H), 6.93 (s, 1H), 3.63 (s, 3H), 2.89 (s, 3H), 2.62 (s,
3H), 2.31 (s, 3H), 2.13 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.23N.sub.6O.sub.2 [M+H].sup.+=439.19, Obtained:
439.58.
##STR00260##
TFA salt yield: 45%. Suzuki
coupling-Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex
--Na.sub.2CO.sub.3 (2M). .sup.1H NMR (300 MHz, MeOD-d4): 9.30 (d,
J=4.44 Hz, 1H), 8.37 (d, J=2.01 Hz, 1H), 8.03 (d, J=4.44 Hz, 1H),
7.84 (d, J=9.00 Hz, 1H), 7.68 (dd, J=9.00, 2.07 Hz, 1H), 7.55 (s,
1H), 6.25 (s, 1H), 3.27 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07
(s, 3H). ESI-MS calculated for
C.sub.26H.sub.21.sup.35ClN.sub.5O.sub.2 [M+H].sup.+=470.14,
Obtained: 470.83.
##STR00261##
The preparation of 2-methoxy-4-bromoquinoline has been reported in
patent WO 2010/030722 and the procedures in the literature were
followed. 2,4-Dibromoquinoline (1.8 g) and sodium methoxide (25% in
MeOH, 1.28 g) were dissolved in anhydrous MeOH (10 mL). The
reaction was heated at reflux for 1 h. The reaction was cooled to
room temperature and MeOH was removed on a rotary evaporator. The
remaining residue was purified by flash column chromatography to
furnish 2-methoxy-4-bromoquinolin in 0.715 g (48% yield).
2-Methoxy-4-bromoquinoline (357 mg) was dissolved in anhydrous THF
(15 mL) and cooled down to -78.degree. C. in a dry ice-ethanol
bath. BuLi (2.5 M in THF, 1 mL) was slowly added via a syringe and
the mixture was stirred at -78.degree. C. for 10 min before
addition of 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(502 mg). The reaction was stirred for another 3 h before quenching
with saturated aqueous NH.sub.4Cl solution. The aqueous layer was
extracted with ethyl acetate and the combined organic layers was
washed with brine and dried over anhydrous sodium sulfate. The
residue was purified by flash column chromatography to furnish the
desired boronate CD215 in 0.35 g (82% yield). .sup.1H NMR (300 MHz,
CDCl.sub.3): 8.52 (d, J=7.51 Hz, 1H), 7.86 (d, J=7.75 Hz, 1H), 7.60
(t, J=8.12 Hz, 1H), 7.41 (s, 1H), 7.40 (t, J=7.58 Hz, 1H), 4.07 (s,
3H), 1.40 (s, 12H).
Suzuki coupling of S13 and CD215 furnished Cpd. No. 89-TFA salt in
35% yield under Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex
--Na.sub.2CO.sub.3 (2M) condition.
##STR00262##
.sup.1H NMR (300 MHz, MeOD-d4): 8.11 (d, J=8.44 Hz, 1H), 7.83 (ddd,
J=8.39, 7.05, 1.30 Hz, 1H), 7.63 (d, J=8.29 Hz, 1H), 7.54 (s, 1H),
7.49 (s, 1H), 7.44 (t, J=7.63 Hz, 1H), 6.27 (s, 1H), 4.20 (s, 3H),
3.21 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.27H.sub.24N.sub.5O.sub.3 [M+H].sup.+=466.19,
Obtained: 466.68.
##STR00263##
4-Bromo-6-chloro-quinoline (500 mg) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.04 g) were
dissolved in anhydrous THF (20 mL) and the mixture was cooled to
-78.degree. C. in a dry ice-ethanol bath. BuLi (2.5 M in THF, 1.2
mL) was slowly added via a syringe and the reaction was stirred for
another 3 h at -78.degree. C. before quenching with saturated
aqueous NH.sub.4Cl solution. The aqueous layer was extracted with
ethyl acetate and the combined organic layers was washed with brine
and dried over anhydrous sodium sulfate. The residue was purified
by flash column chromatography to furnish the desired
6-chloro-quinoline-4-boronic acid pinacol ester CD224 in 0.37 g
(67% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.87 (d, J=4.08 Hz,
1H), 8.62 (d, J=2.14 Hz, 1H), 8.02 (d, J=8.98 Hz, 1H), 7.85 (d,
J=4.08 Hz, 1H), 7.60 (dd, J=8.97, 2.15 Hz, 1H), 1.40 (s, 12H).
ESI-MS calculated for C.sub.15H.sub.18B.sup.35ClNO.sub.2
[M+H].sup.+=290.11, observed: 290.56.
Suzuki coupling of S13 and CD224 furnished Cpd. No. 90-TFA salt in
44% yield under Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex
--Na.sub.2CO.sub.3 (2M) condition.
##STR00264##
.sup.1H NMR (300 MHz, MeOD-d4): 12.18 (s, broad, 1H), 9.26 (s, 1H),
8.35 (d, J=8.79 Hz, 1H), 7.97 (s, 1H), 7.82 (d, J=8.89 Hz, 1H),
7.76 (s, 1H), 7.26 (s, 1H), 6.24 (s, 1H), 3.46 (s, 3H), 2.96 (s,
3H), 2.25 (s, 3H), 2.09 (s, 3H). .sup.13C NMR (75 MHz, MeOD-d4):
166.78, 160.20, 159.65, 155.43, 154.08, 150.63, 148.94, 144.89,
140.19, 135.71, 134.25, 132.90, 130.27, 125.55, 124.19, 123.11,
118.29, 116.30, 113.03, 112.29, 103.89, 55.47, 23.25, 11.72, 10.69.
ESI-MS calculated for C.sub.26H.sub.21.sup.35ClN.sub.5O.sub.2
[M+H].sup.+=470.14, Obtained: 470.60.
7-Fluoroquinoline-4-boronic acid pinacol ester CD223 was prepared
from 4-bromo-7-fluoro-quinoline in 75% yield using the same method
for preparation of CD224. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.81
(d, J=4.20 Hz, 1H), 8.59 (dd, J=9.26, 6.29 Hz, 1H), 7.75 (d, J=4.19
Hz, 1H), 7.68 (dd, J=10.07, 2.62 Hz, 1H), 7.27 (ddd, J=8.82, 8.70,
2.55 Hz, 1H), 1.33 (s, 12H). .sup.13C NMR (75 MHz, CDCl.sub.3):
164.31, 161.00, 150.24, 148.68 (d, J.sub.C--F=12.54 Hz), 130.76 (d,
J.sub.C--F=9.51 Hz), 128.20, 128.03 (d, J.sub.C--F=1.95 Hz), 117.20
(d, J.sub.C--F=24.54 Hz), 112.81 (d, J.sub.C--F=20.13), 84.71,
24.93. ESI-MS calculated for C.sub.15H.sub.18BFNO.sub.2
[M+H].sup.+=274.14, observed: 274.75.
Suzuki coupling of 7-fluoroquinoline-4-boronic acid pinacol ester
(CD223) and S13 furnished Cpd. No. 91-TFA salt in 31% under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
condition.
##STR00265##
.sup.1H NMR (300 MHz, MeOD-d4): 9.26 (d, J=4.46 Hz, 1H), 8.02-7.84
(m, 3H), 7.55-7.46 (m, 1H), 7.49 (s, 1H), 6.24 (s, 1H), 3.26 (s,
3H), 2.95 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated
for C.sub.26H.sub.21FN.sub.5O.sub.2 [M+H].sup.+=454.17, Obtained:
454.42.
6-Fluoroquinoline-4-boronic acid pinacol ester CD234 was prepared
from 4-bromo-6-fluoro-quinoline in 85% yield using the same method
for preparation of CD224. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.85
(d, J=4.09 Hz, 1H), 8.30 (dd, J=10.48, 2.69 Hz, 1H), 8.07 (dd,
J=9.20, 5.71 Hz, 1H), 7.84 (d, J=4.02 Hz, 1H), 7.44 (ddd, J=9.46,
9.25, 2.76 Hz, 1H), 1.39 (s, 12H). ESI-MS calculated for
C.sub.15H.sub.18BFNO.sub.2 [M+H].sup.+=274.14, observed:
274.67.
Suzuki coupling of 6-fluoroquinoline-4-boronic acid pinacol ester
(CD234) and S13 furnished Cpd. No. 92-TFA salt in 47% under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
condition.
##STR00266##
.sup.1H NMR (300 MHz, MeOD-d4): 9.23 (d, J=4.44 Hz, 1H), 8.39 (dd,
J=9.33, 5.33 Hz, 1H), 8.01 (d, J=4.42 Hz, 1H), 7.80 (ddd, J=9.25,
8.32, 2.78 Hz, 1H), 7.56-7.48 (m, 1H), 7.52 (s, 1H), 6.28 (s, 1H),
3.27 (s, 3H), 2.97 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.21FN.sub.5O.sub.2 [M+H].sup.+=454.17,
Obtained: 454.44.
2-Isopropylpyridine-4-boronic acid pinacol ester CD263 was prepared
from 4-bromo-2-isopropylpyridine in 30% yield using the same method
for preparation of CD224. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.55
(d, J=5.62 Hz, 1H), 7.51 (s, 1H), 7.43 (d, J=5.60 Hz, 1H), 3.07
(septet, J=6.92 Hz, 1H), 1.35 (s, 12H), 1.31 (d, J=6.93 Hz,
6H).
Suzuki coupling of 2-isopropylpyridine-4-boronic acid pinacol ester
(CD263) and S13 furnished Cpd. No. 93-TFA salt in 46% yield under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
condition.
##STR00267##
.sup.1H NMR (300 MHz, MeOD-d4): 9.00 (d, J=5.61 Hz, 1H), 8.29 (d,
J=0.90 Hz, 1H), 8.22 (dd, J=5.63, 1.65 Hz, 1H), 7.52 (s, 1H), 7.29
(s, 1H), 3.73 (s, 3H), 3.46 (septet, J=6.95 Hz, 1H), 2.92 (s, 3H),
2.31 (s, 3H), 2.14 (s, 3H), 1.51 (d, J=6.95 Hz, 6H). .sup.13C NMR
(75 MHz, MeOD-d4): 168.16, 167.57, 163.02, 161.14, 158.96, 155.20,
152.54, 149.33, 147.19, 135.98, 124.60, 124.52, 123.63, 120.32,
116.89, 114.70, 111.33, 105.43, 56.67, 36.31, 24.24, 22.55, 11.68,
10.81. ESI-MS calculated for C.sub.25H.sub.26N.sub.5O.sub.2
[M+H].sup.+=428.21, Obtained: 428.75.
2-Methylpyridine-4-boronic acid pinacol ester is commercially
available from Small Molecules Inc. It has also been prepared from
following procedures: 4-Bromo-2-methylpyridine (1.0 g),
Bis(pinacolato)diboron (1.4 g), potassium acetate (1.35 g), and
anhydrous dioxane (30 mL) was mixed in a round-bottom flask. The
system was degassed to remove oxygen and Pd(dppf)Cl.sub.2 (35 mg)
was added in one portion. The system was degassed again and heated
at 100.degree. C. for 12 h. The reaction was cooled to room
temperature and black precipitate was removed by filtration. The
solvent was removed on a rotary evaporator and the residue was
purified by flash column to furnish 2-methylpyridine-4-boronic acid
pinacol ester.
Suzuki coupling of 2-methylpyridine-4-boronic acid pinacol ester
and S13 furnished Cpd. No. 94-TFA salt in 27% yield under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
condition.
##STR00268##
.sup.1H NMR (300 MHz, MeOD-d4): 8.98 (d, J=5.85 Hz, 1H), 8.38 (s,
1H), 8.30 (d, J=5.62 Hz, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 3.76 (s,
3H), 3.30 (s, 3H), 2.89 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H). ESI-MS
calculated for C.sub.23H.sub.22N.sub.5O.sub.2 [M+H].sup.+=400.18,
Obtained: 400.52.
3-Methylpyridine-4-boronic acid pinacol ester CD275 was prepared
from 4-bromo-3-methylpyridine using the same method for the
preparation of 2-methylpyridine-4-boronic acid pinacol ester in 54%
yield. ESI-MS calculated for C.sub.12H.sub.19BNO.sub.2
[M+H].sup.+=220.15, Obtained: 220.72.
Suzuki coupling of 3-methylpyridine-4-boronic acid pinacol ester
and S13 furnished Cpd. No. 95-TFA salt in 27% yield under
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex --Na.sub.2CO.sub.3 (2M)
condition.
##STR00269##
.sup.1H NMR (300 MHz, MeOD-d.sub.4): 8.98 (s, 1H), 8.89 (d, J=5.25
Hz, 1H), 7.98 (d, J=5.32 Hz, 1H), 7.52 (s, 1H), 6.73 (s, 1H), 3.61
(s, 3H), 2.92 (s, 3H), 2.41 (s, 3H), 2.30 (s, 3H), 2.12 (s, 3H).
ESI-MS calculated for C.sub.23H.sub.22N.sub.5O.sub.2
[M+H].sup.+=400.18, Obtained: 400.58.
##STR00270##
TFA salt yield: 29% Suzuki
coupling-Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2
complex-Na.sub.2CO.sub.3 (2M). The boronic acid required is
commercially available. .sup.1H NMR (300 MHz, MeOD-d.sub.4): 9.41
(s, 1H), 8.35 (s, 1H), 7.38 (s, 1H), 7.12 (s, 1H), 7.02 (s, 5H),
5.72 (s, 2H), 3.75 (s, 3H), 2.84 (s, 3H), 2.30 (s, 3H), 2.14 (s,
3H). ESI-MS calculated for C.sub.27H.sub.25N.sub.6O.sub.2
[M+H].sup.+=465.20, Obtained: 465.58.
The preparation of 4-nitro-5,6,7,8-tetrahydroquinoline-N-oxide has
been previously reported in WO02076979 and the same procedure was
followed.
##STR00271##
5,6,7,8-Tetrahydroquinoline (5.2 g) was dissolved in 100 mL
anhydrous THF and the solution was cooled with ice-water bath.
m-CPBA (10.8 g) was added in small portions and the mixture was
stirred at 0.degree. C. for 1 h. THF was then removed on a rotary
evaporator and the residue was dissolved in CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 solution was washed with NaOH (2 N, 20 mL) and
citric acid (10%, 40 mL) and dried over anhydrous sodium sulfate.
The solvent was removed on a rotary evaporator and the residue of
5,6,7,8-tetrahydroquinoline-N-oxide was used for the next step
without purification.
To a round bottom flask containing
5,6,7,8-tetrahydroquinoline-N-oxide was added a mixture of
HNO.sub.3--H.sub.2SO.sub.4 (10 mL (90%): 14 mL (98%)) at 0.degree.
C. The mixture was then heated at 80.degree. C. for 2 h and was
then poured onto ice cubes. The aqueous layer was extracted with
CH.sub.2Cl.sub.2 to furnish
4-nitro-5,6,7,8-tetrahydroquinoline-N-oxide. The crude material was
used without further purification.
The crude 4-nitro-5,6,7,8-tetrahydroquinoline-N-oxide was dissolved
in acetic acid (40 mL) and zinc powder (20.8 g) was slowly added at
room temperature and the mixture was heated at 80.degree. C. for 4
h. The precipitate was removed by filtration and washed with acetic
acid. The combined acetic acid solution was concentrated on a
rotary evaporator and was neutralized by aqueous NaOH solution. The
aqueous solution was extracted by chloroform (50 mL.times.8) and
the combined organic phase was dried over anhydrous sodium sulfate.
The solvent was removed on a rotary evaporator and the residue was
purified by flash column chromatogram to furnish
4-amino-5,6,7,8-tetrahydroquinoline in 1.4 g (23% over three
steps).
4-Amino-5,6,7,8-tetrahydroquinoline (1.4 g) was dissolved in 48%
HBr (6.7 mL) and the solution was cooled to -10.degree. C. To this
solution, Br.sub.2 was added via a syringe followed by slow
addition of NaNO.sub.2 (3.3 g) in 4 mL water and the reaction
mixture was warm up to room temperature and stirred at room
temperature for 1 h. The reaction mixture was then poured onto ice
and the pH was adjusted=9 using aqueous sodium hydroxide solution.
The aqueous layer was extracted with ethyl acetate and combined
organic layer was dried over anhydrous sodium sulfate. Removal of
solvent on a rotary evaporator and the remaining residue was
purified by flash column chromatogram to furnish
4-bromo-5,6,7,8-tetrahydroquinoline in 1.00 g (47% yield).
4-Bromo-5,6,7,8-tetrahydroquinoline (0.5 g), bis(pinacolato)diboron
(1 g), potassium acetate (735 mg), and anhydrous dioxane (20 mL)
were placed in a round-bottom flask. The system was degassed to
remove oxygen followed by the addition of Pd(dffp)Cl.sub.2 (176 mg)
in one portion. The system was degassed again and the reaction was
heated at 100.degree. C. for 12 h. The reaction was cooled to room
temperature and black precipitate was removed by filtration. The
solvent was removed on a rotary evaporator and the residue was
purified by flash column chromatogram to furnish
5,6,7,8-tetrahydroquinoline-4-boronic acid pinacol ester CD292 in
0.18 g (28% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.32 (d,
J=4.58 Hz, 1H), 7.36 (d, J=4.59 Hz, 1H), 2.98 (t, J=6.03 Hz, 2H),
2.92 (t, J=6.09 Hz, 2H), 1.90-1.72 (m, 4H), 1.33 (s, 12H). ESI-MS
calculated for C.sub.15H.sub.23BNO.sub.2 [M+H].sup.+=260.18,
Obtained: 260.33.
Suzuki coupling of 5,6,7,8-tetrahydroquinoline-4-boronic acid
pinacol ester and S13 furnished Cpd. No. 97-TFA salt in 33% yield
under Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 complex-Na.sub.2CO.sub.3
(2M) condition.
##STR00272##
.sup.1H NMR (300 MHz, MeOD-d4): 8.86 (d, J=5.76 Hz, 1H), 8.02 (d,
J=5.76 Hz, 1H), 7.50 (s, 1H), 6.82 (s, 1H), 3.66 (s, 3H), 3.00-2.80
(m, 1H), 2.88 (s, 3H), 2.80-2.50 (m, 1H), 2.30 (s, 3H), 2.12 (s,
3H), 2.10-2.00 (m, 2H), 2.00-1.70 (m, 2H). .sup.13C NMR (75 MHz,
MeOD-d4): 168.13, 163.57, 161.14, 158.66, 157.33, 155.40, 151.94,
150.93, 142.17, 137.35, 135.78, 125.61, 123.39, 120.14, 116.89,
114.71, 111.32, 104.82, 56.69, 49.21, 30.02, 27.15, 24.65, 22.51,
22.21, 11.66, 10.78. ESI-MS calculated for
C.sub.26H.sub.26N.sub.5O.sub.2 [M+H].sup.+=440.21, Obtained:
440.67
##STR00273##
Cpd. No. 98 was synthesized from S13 and
8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-[1,4]dioxino[-
2,3-b]pyridine using Suzuki coupling condition
[Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification yielded
the Cpd. No. 98-TFA salt in 25% yield. .sup.1H NMR (300 MHz,
MeOD-d4): 8.12 (d, J=5.09 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J=5.09
Hz, 1H), 7.15 (s, 1H), 4.65-4.55 (m, 2H), 4.44-4.35 (m, 2H), 3.72
(s, 3H), 2.97 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H). ESI-MS
calculated for C.sub.24H.sub.22N.sub.5O.sub.4 [M+H].sup.+=444.17,
Obtained: 444.46.
##STR00274##
Cpd. No. 99 was synthesized from S13 and
3-(N,N-dimethylamino)phenylboronic acid using Suzuki coupling
condition [Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification yielded
the Cpd. No. 99-TFA salt in 50% yield. .sup.1H NMR (300 MHz,
MeOD-d4): 7.72-7.62 (m, 1H), 7.54 (s, 1H), 7.42 (s, 1H), 7.40-7.30
(m, 2H), 7.28 (dd, J=8.47, 2.05 Hz, 1H), 3.68 (s, 3H), 3.12 (s,
6H), 2.96 (s, 3H), 2.30 (s, 3H), 2.13 (s, 3H). ESI-MS calculated
for C.sub.25H.sub.26N.sub.5O.sub.2 [M+H].sup.+=428.21, Obtained:
428.58.
##STR00275##
CD303,
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,8-naphthyridine,
was synthesized following the method shown in the above scheme.
4-Bromo-1,8-naphthyridine (400 mg), bis(pinacolato)diboron (1.0 g),
and KOAc (600 mg) were placed in a round-bottom flask equipped with
a magnetic stirring bar Anhydrous-1,4-dioxane (20 mL) was added and
the mixture was degassed for 5 min to remove oxygen.
Pd(dppf)Cl.sub.2 (140 mg) was added and the system was again
degassed and followed by refilling nitrogen. The mixture was heated
at 100.degree. C. for overnight (>12 h). The reaction was cooled
to room temperature and filtered. The volatile components were
removed on a rotary evaporator and the residue was purified in a
preparative HPLC to yield CD303-TFA salt in 0.38 g. .sup.1H NMR
(300 MHz, MeOD-d4): 9.67 (d, J=8.48 Hz, 1H), 9.30 (t, J=5.11 Hz,
2H), 8.33 (d, J=4.36 Hz, 1H), 8.10 (dd, J=8.36, 4.89 Hz, 1H), 1.46
(s, 12H). ESI-MS calculated for C.sub.14H.sub.18BN.sub.2O.sub.2
[M+H].sup.+=257.15, obtained: 257.44.
##STR00276##
Cpd. No. 100 was synthesized from S13 and
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,8-naphthyridine-TFA
salt using Suzuki coupling condition
[Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification yielded
the Cpd. No. 100-TFA salt in 5% yield. .sup.1H NMR (300 MHz,
MeOD-d4): 9.50 (d, J=4.40 Hz, 1H), 9.30 (d, J=4.29 Hz, 1H), 8.49
(dd, J=8.46, 1.76 Hz, 1H), 8.21 (d, J=4.48 Hz, 1H), 7.79 (dd,
J=8.47, 4.38 Hz, 1H), 7.53 (s, 1H), 6.39 (s, 1H), 2.98 (s, 3H),
2.27 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.21N.sub.6O.sub.2 [M+H].sup.+=437.17, Obtained:
437.42.
##STR00277##
N.sup.1,N.sup.1-Dimethyl-N.sup.2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborola-
n-2-yl)quinolin-2-yl)ethane-1,2-diamine was synthesized following
the method shown in the above scheme
Step 1: synthesis of CD298. 2,4-Dibromoquinoline (572 mg),
N.sup.1,N.sup.1-dimethylethane-1,2-diamine (240 mg),
Cs.sub.2CO.sub.3 (652 mg), and DMSO (4 mL) were placed in a sealed
tube. The mixture was heated at 170.degree. C. for 12 h. The
mixture was cooled to room temperature and purified on a reverse
phase preparative HPLC. The desired product CD298-TFA salt was
isolated in 0.47 g (57% yield). The 4-amination regio-isomer was
also isolated in ca. 40% yield. Free amine CD298 was also purified
by flash column chromatography but in a compromised yield. The
structure of CD298 was confirmed by comparing .sup.1H NMR data of
2-bromo-N-methylquinolin-4-amine (Chemistry of heterocyclic
compounds, vol 34, No. 7, 1998, page 837), 2-bromoquinolin-4-amine
(J Med Chem 2009, 52, 926-931), and 4-bromoquinolin-2-amine
(biochemistry, 2004, 43, 1440-1448). .sup.1H NMR (300 MHz,
CDCl.sub.3, free amine): 7.93 (d, J=8.23 Hz, 1H), 7.65 (d, J=8.36
Hz, 1H), 7.54 (ddd, J=8.36, 6.96, 1.40 Hz, 1H), 7.25 (ddd, J=8.14,
6.91, 1.17 Hz, 1H), 6.98 (s, 1H), 5.40 (broad, 1H), 3.54 (dd,
J=11.33, 5.25 Hz, 2H), 2.56 (t, J=5.81 Hz, 1H), 2.26 (s, 6H).
ESI-MS calculated for C.sub.13H.sub.17.sup.79BrN.sub.3
[M+H].sup.+=294.06, obtained: 294.83.
Step 2: synthesis of CD302. CD302
[N.sup.1,N.sup.1-dimethyl-N.sup.2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)quinolin-2-yl)ethane-1,2-diamine] was synthesized from
coupling of CD298 and bis(pinacolato)diboron using the same method
for the preparation of CD303
[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,8-naphthyridine].
CD302 was obtained in 90% yield. .sup.1H NMR (300 MHz, MeOD-d4):
8.53 (d, J=7.89 Hz, 1H), 7.91 (d, J=7.94 Hz, 1H), 7.75 (ddd, 8.43,
7.28, 1.26 Hz, 1H), 7.52 (ddd, J=8.39, 7.24, 1.11 Hz, 1H), 7.50 (s,
1H), 4.10 (t, J=6.18 Hz, 2H), 3.58 (t, J=6.18 Hz, 2H), 3.00 (s,
6H), 1.42 (s, 12ESI-MS calculated for
C.sub.19H.sub.29BN.sub.3O.sub.2 [M+H].sup.+=342.24, obtained:
342.50
##STR00278##
Cpd. No. 101 was synthesized from S13 and
N.sup.1,N.sup.1-dimethyl-N.sup.2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-
an-2-yl)quinolin-2-yl)ethane-1,2-diamine using Suzuki coupling
condition [Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification yielded
the Cpd. No. 102-TFA salt in 37% yield. .sup.1H NMR (300 MHz,
MeOD-d4): 8.09 (d, J=8.35 Hz, 1H), 7.88 (t, J=7.31 Hz, 1H), 7.63
(d, J=7.62 Hz, 1H), 7.62 (s, 1H), 7.51 (s, 1H), 7.43 (t, J=7.62 Hz,
1H), 6.49 (s, 1H), 4.15 (t, J=6.05 Hz, 2H), 3.63 (t, J=6.15 Hz,
2H), 3.31 (s, 3H), 3.04 (s, 6H), 2.98 (s, 3H), 2.26 (s, 3H), 2.07
(s, 3H). ESI-MS calculated for C.sub.30H.sub.32N.sub.7O.sub.2
[M+H].sup.+=522.26, Obtained: 522.50.
##STR00279##
Cpd. No. 102 was synthesized from S13 and
(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)boronic acid using Suzuki
coupling condition [Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst
and Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification
yielded the Cpd. No. 102-TFA salt in 28% yield. .sup.1H NMR (300
MHz, MeOD-d4): 7.54 (s, 1H), 7.40-7.20 (m, 3H), 7.17 (s, 1H),
4.45-4.36 (m, 2H), 4.36-4.30 (m, 2H), 3.68 (s, 3H), 2.95 (s, 3H),
2.31 (s, 3H), 2.13 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.23N.sub.4O.sub.4 [M+H].sup.+=443.17, Obtained:
443.44.
##STR00280##
Cpd. No. 103 was synthesized from S13 and
2-isopropoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
using Suzuki coupling condition [Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2
as catalyst and Na.sub.2CO.sub.3 2 M in water as base]. HPLC
purification yielded the Cpd. No. 103-TFA salt in 43% yield.
.sup.1H NMR (300 MHz, MeOD-d4): 8.56 (d, J=5.20 Hz, 1H), 7.55 (s,
1H), 7.47 (dd, J=5.22, 1.40 Hz, 1H), 7.34 (d, J=3.55 Hz, 1H), 5.49
(septet, J=6.17 Hz, 1H), 3.73 (s, 3H), 2.95 (s, 3H), 2.31 (s, 3H),
2.14 (s, 3H), 1.41 (d, J=6.17 Hz, 6H). ESI-MS calculated for
C.sub.25H.sub.26N.sub.5O.sub.3 [M+H].sup.+=444.20, Obtained:
444.40.
##STR00281##
Suzuki coupling of S13 and
2-(tert-butoxy)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
[condition: Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base] afforded a mixture of Cpd.
No. 104 and tert-Bu ether form of Cpd. No. 104. The mixture was
treated with TFA followed by HPLC purification yielded the Cpd. No.
104-TFA salt as the major product. .sup.1H NMR (300 MHz, MeOD-d4):
7.87 (d, J=6.71 Hz, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.12 (s, 1H),
6.88 (dd, J=6.71, 1.69 Hz, 1H), 3.80 (s, 3H), 2.94 (s, 3H), 2.32
(s, 3H), 2.14 (s, 3H). ESI-MS calculated for
C.sub.22H.sub.20N.sub.5O.sub.3 [M+H].sup.+=402.16, Obtained:
402.67.
##STR00282##
Cpd. No. 105 was synthesized from S13 and
4-(N,N-Dimethylamino)phenylboronic acid using Suzuki coupling
condition [Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 as catalyst and
Na.sub.2CO.sub.3 2 M in water as base]. HPLC purification yielded
the Cpd. No. 105-TFA salt in 31% yield. .sup.1H NMR (300 MHz,
MeOD-d4): 7.94 (d, J=9.0 Hz, 2H), 7.64 (s, 1H), 7.51 (s, 1H), 7.08
(d, J=9.0 Hz, 2H), 3.77 (s, 3H), 3.17 (s, 3H), 2.91 (s, 3H), 2.32
(s, 3H), 2.14 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.26N.sub.5O.sub.2 [M+H].sup.+=428.21, Obtained:
428.42.
CD278 (25 mg, CF.sub.3CO.sub.2H salt) was dissolved in THF (15 mL).
The solution was degassed to remove oxygen and 10% Pd on activated
charcoal (20 mg) was added. A hydrogen balloon was applied to the
reaction system and the reaction was stopped after 12 h.
Pd-charcoal was filtered off and solvent was removed on a rotary
evaporator. The residues were purified by reverse phase HPLC to
yield the desired product Cpd. No. 106 in 17 mg (81% yield) as a
salt of CF.sub.3CO.sub.2H.
##STR00283##
.sup.1H NMR (300 MHz, MeOD-d4): 8.94 (s, 1H), 8.48 (s, 1H), 8.32
(s, 1H), 7.48 (s, 1H), 3.94 (s, 3H), 2.92 (s, 3H), 2.34 (s, 3H),
2.17 (s, 3H). ESI-MS calculated for C.sub.20H.sub.19N.sub.6O.sub.2
[M+H].sup.+=375.16, Obtained: 375.83.
CD281 (10 mg, CF.sub.3CO.sub.2H salt), iodobenzene (204 mg),
Cs.sub.2CO.sub.3 (650 mg), proline (22 mg), CuI (40 mg), and DMF (5
mL) were placed in a round-bottom flask. The mixture was degassed
to remove oxygen and then heated up at 120.degree. C. for 12 h. The
reaction mixture was quenched with water and the aqueous layer was
extracted with ethyl acetate (50 mL.times.3). The combined organic
layers were washed with brine and dried over anhydrous sodium
sulfate. The solvent was removed on a rotary evaporator and the
residue was purified by a phase HPLC. The desired product Cpd. No.
107 was obtained as TFA salt in 10 mg (19% yield).
##STR00284##
.sup.1H NMR (300 MHz, MeOD-d4): 9.36 (s, 1H), 8.92 (s, 1H), 8.73
(s, 1H), 7.80-7.52 (m, 10H), 7.25 (s, 1H), 4.02 (s, 3H), 2.90 (s,
3H), 2.28 (s, 3H), 2.12 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.27N.sub.6O.sub.2 [M+H].sup.+=527.22, Obtained:
527.67.
CD281 (TFA salt, 15 mg), PhB(OH).sub.2 (36 mg), Cu(OAc).sub.2 (46
mg) and anhydrous molecular sieve 4A (250 mg) were placed in a
round-bottom flask. Pyridine (0.05 mL) and CH.sub.2Cl.sub.2 (5 mL)
was added via syringes. An oxygen balloon was applied to the
reaction mixture and the reaction was stirred at room temperature
for 2 days. The reaction was filtered through a pad of Celite.RTM.
and the Celite.RTM. was washed with methanol. The organic layers
were combined and the solvent was removed on a rotary evaporator.
The remaining residue was purified by reverse phase HPLC to yield
Cpd. No. 108 (4 mg, 22%), Cpd. No. 109 (2 mg, 12%), and Cpd. No.
107 (4 mg, 25%), with Cpd. Nos. 108 and 109 in the form of
TFA-salt.
##STR00285##
.sup.1H NMR (300 MHz, MeOD-d4): 9.17 (s, 1H), 8.82 (s, 1H), 8.67
(s, 1H), 7.77 (d, J=8.05 Hz, 2H), 7.65 (t, J=7.76 Hz, 2H), 7.56 (d,
J=7.33 Hz, 1H), 7.48 (s, 1H), 3.98 (s, 3H), 2.94 (s, 3H), 2.35 (s,
3H), 2.18 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.23N.sub.6O.sub.2 [M+H].sup.+=451.19, Obtained:
451.50.
##STR00286##
.sup.1H NMR (300 MHz, MeOD-d4): 9.16 (s, 1H), 8.33 (s, 1H),
7.75-7.55 (m, 6H), 7.44 (s, 4H), 7.21 (s, 1H), 3.88 (s, 3H), 2.50
(s, 3H), 2.25 (s, 3H), 2.10 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.27N.sub.6O.sub.2 [M+H].sup.+=527.22, Obtained:
527.58.
S13 (34 mg), benzimidazole (24 mg), Cs.sub.2CO.sub.3 (190 mg), and
DMSO (4 mL) were placed in a sealed tube equipped with a magnetic
stirring bar. The reaction mixture was heated up at 170.degree. C.
for 12 h. The reaction mixture was diluted with water and the
aqueous layer was extracted with ethyl acetate (50 mL.times.2). The
combined organic layers were dried over anhydrous sodium sulfate
and the solvent was removed on a rotary evaporator. The residue was
purified by a reverse phase HPLC to yield Cpd. No. 110 as a salt of
CF.sub.3CO.sub.2H (18 mg, 33% yield).
##STR00287##
.sup.1H NMR (300 MHz, MeOD-d4): 9.16 (s, 1H). 7.95 (d, J=7.91 Hz,
1H), 7.59-7.44 (m, 3H), 7.43 (s, 1H), 6.68 (s, 1H), 3.40 (s, 3H),
2.83 (s, 3H), 2.29 (s, 3H), 2.11 (s, 3H). ESI-MS calculated for
C.sub.24H.sub.21N.sub.6O.sub.2 [M+H].sup.+=425.17, Obtained:
425.32.
Cpd. No. 111 was prepared from S13 and 2-methylbenzimidazole in 5%
yield as a salt of CF.sub.3CO.sub.2H using the same condensation
method for the preparation of Cpd. No. 110.
##STR00288##
TFA salt .sup.1H NMR (300 MHz, MeOD-d4): 7.89 (d, J=8.20 Hz, 1H),
7.52 (t, J=7.82 Hz, 1H), 7.44 (s, 1H), 7.39 (t, J=7.82 Hz, 1H),
7.18 (d, J=8.57 Hz, 1H), 6.23 (s, 1H), 3.25 (s, 3H), 2.88 (s, 3H),
2.78 (s, 3H), 2.28 (s, 3H), 2.09 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.23N.sub.6O.sub.2 [M+H].sup.+=439.19, Obtained:
439.40.
##STR00289## ##STR00290##
S6 (400 mg) was dissolved in isobutyronitrile (2 mL). HCl gas was
bubbled into the solution for 40 min and the solution was heated at
90.degree. C. for 3 h. The solvent was concentrated in vacuum and
the residue was dissolved in ethanol (40 mL). NaOH (10%, 30 mL) was
added to the ethanol solution and the mixture was heated at reflux
for overnight. The solution was cooled to room temperature and
concentrated in vacuum. Ethyl acetate (20 mL) was added followed by
aqueous HCl solution to set pH=4-5. The precipitate was collected
by filtration and the residue was washed with diethyl ether to
furnish CD171 in 0.26 g. .sup.1H NMR (300 MHz, DMSO-d6): 12.05 (s,
1H), 12.00 (s, 1H), 7.54 (s, 1H), 7.18 (s, 1H), 3.81 (s, 3H), 2.97
(septet, J=6.75 Hz, 1H), 2.26 (s, 3H), 2.06 (s, 3H), 1.25 (d,
J=6.80 Hz, 6H).
CD171 (0.26 g) was mixed with phosphorus(V) oxychloride (5 mL) and
heated at 90.degree. C. for 6 h. The mixture was concentrated in
vacuum and neutralized with excess aqueous NaHCO.sub.3 saturated
solution. Ethyl acetate (30 mL) was added and the precipitate was
collected by filtration. The solid residue was washed with diethyl
ether to furnish CD177 in 120 mg (43% yield). .sup.1H NMR (300 MHz,
DMSO-d6): 12.52 (s, 1H), 7.79 (s, 1H), 7.38 (s, 1H), 3.88 (s, 3H),
3.19 (septet, J=6.88 Hz, 1H), 2.28 (s, 3H), 2.09 (s, 3H), 1.33 (d,
J=6.88 Hz, 6H).
Suzuki coupling of tert-butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate and CD177 and deprotection of Boc group in
TFA-CH.sub.2Cl.sub.2 provided Cpd. No. 112 in 36% yield as a salt
of CF.sub.3CO.sub.2H using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3
condition.
##STR00291##
.sup.1H NMR (300 MHz, MeOD-d4): 11.91 (s, 1H), 7.59 (d, J=8.10 Hz,
1H), 7.53 (s, 1H), 7.31 (ddd, J=8.16, 6.70, 1.33 Hz, 1H), 7.28-7.14
(m, 2H), 6.76 (s, 1H), 3.50 (septet, J=6.77 Hz, 1H), 3.00 (s, 3H),
2.66 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H), 1.56 (d, J=6.77 Hz, 3H),
1.55 (d, J=6.76 Hz, 3H). ESI-MS calculated for
C.sub.28H.sub.28N.sub.5O.sub.2 [M+H].sup.+=466.22, Obtained:
466.58
##STR00292## ##STR00293##
S6 (300 mg), tetrahydropyranyl-4-carbonitrile (330 mg), and dioxane
(10 mL) were placed in a round-bottom flask. HCl gas was bubbled
into the solution for 40 min and the solution was heated at
80.degree. C. for 5 h. The solvent was concentrated in vacuum and
the residue was dissolved in ethanol (30 mL). NaOH (10%, 30 mL) was
added to the ethanol solution and the mixture was heated at reflux
for 12 h. The solution was cooled to room temperature and
concentrated in vacuum. Ethyl acetate (20 mL) was added followed by
addition of aqueous HCl solution to set pH=4-5. The precipitate was
collected by filtration and the residue was washed with diethyl
ether to furnish CD188 in 0.12 g (33% yield). ESI-MS calculated for
C.sub.21H.sub.23N.sub.4O.sub.4 [M+H].sup.+=395.17, Obtained:
395.58.
CD188 (0.12 g) was mixed with phosphorus(V) oxychloride (10 mL) and
heated at 90.degree. C. for 6 h. The mixture was concentrated in
vacuum and neutralized with excess aqueous NaHCO.sub.3 saturated
solution. Ethyl acetate (20 mL) was added and the precipitate was
collected by filtration. The solid residue was washed with diethyl
ether to furnish CD197 in 80 mg (63% yield).
Suzuki coupling of quinoline-4-boronic acid pinacol ester and CD197
furnished Cpd. No. 113-TFA salt in 8% yield using
Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 condition.
##STR00294##
.sup.1H NMR (300 MHz, MeOD-d4): 9.31 (d, J=4.76 Hz, 1H), 8.36 (d,
J=8.27 Hz, 1H), 8.10 (d, J=4.75 Hz, 1H), 8.05 (ddd, J=8.44, 6.91,
1.31 Hz, 1H), 7.92 (d, J=7.91 Hz, 1H), 7.79-7.70 (m, 1H), 7.48 (s,
1H), 6.29 (s, 1H), 4.12 (d, J=14.04 Hz, 2H), 3.64 (td, J=11.48,
2.27 Hz, 2H), 3.50-3.30 (m, 1H), 3.24 (s, 3H), 2.27 (s, 3H),
2.25-2.14 (m, 2H), 2.14-2.03 (m, 2H), 2.08 (s, 3H). ESI-MS
calculated for C.sub.30H.sub.28N.sub.5O.sub.3 [M+H].sup.+=506.22,
Obtained: 506.33.
##STR00295## ##STR00296##
##STR00297##
To a mixture of
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(H1) (12.2 g, 54.6 mmol, 2 equiv), 4-bromo-1-fluoro-2-nitrobenzene
(H2) (6 g, 27.3 mmol, 1 equiv) and K.sub.2CO.sub.3 (11.3 g, 81.9
mmol, 3.0 equiv), 1,2-dimethoxyethane (60 mL) and water (40 mL)
were added at room temperature. The mixture was purged with
nitrogen before Pd(PPh.sub.3).sub.4 (1.6 g, 1.4 mmol, 0.05 equiv)
was added in one portion. The reaction mixture was purged with
nitrogen and refluxed at 90.degree. C. overnight. The aqueous layer
was extracted with ethyl acetate and combined organic layers were
washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The residue was purified over flash column
chromatography furnishing 5.5 g (23.1 mmol) of the intermediate
HK-06-203 as a bright yellow solid (85% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 7.94 (dd, J=2.3 Hz, J=7.0 Hz, 1H), 7.57-7.50
(m, 1H), 7.39 (dd, J=8.7 Hz, J=10.4 Hz, 1H), 2.42 (s, 3H), 2.27 (s,
3H).
##STR00298##
To a suspension of NaH (1.8 g, 60% in mineral oil, 46 mmol, 2.0
equiv) in dry DMF (40 mL), ethyl cyanoacetate (3.9 g, 34.5 mmol,
1.5 equiv) was added dropwise at 0.degree. C. and the reaction was
stirred at room temperature for 30 min. The mixture was cooled to
0.degree. C., anhydrous DMF solution (20 mL) of HK-06-203 (5.45 g,
23 mmol, 1.0 equiv) was added via a syringe. The reaction mixture
was allowed to warm up to room temperature and was stirred
overnight. Ethyl acetate (50 ml) and MeOH (20 ml) were added to the
reaction mixture, and pH was adjusted to 2-3 with aqueous 2 N HCl.
The volatile components were removed on a rotary evaporator and the
residue was purified over a flash column chromatography furnishing
6.9 g (21 mmol) of the intermediate HK-06-204 as bright yellow oil
(91% yield). .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.12 (d, J=1.6 Hz,
1H), 7.86 (d, J=8.0 Hz, 1H), 7.66 (dd, J=1.6 Hz, J=8.0 Hz, 1H),
5.69 (s, 1H), 4.34 (q, J=7.1 Hz, 2H), 2.48 (s, 3H), 2.32 (s, 3H),
1.35 (t, J=7.1 Hz, 3H).
##STR00299##
Acetic acid (23 mL) solution of HK-06-204 (2.5 g, 7.5 mmol, 1.0
equiv) at 85.degree. C., zinc powder (1.21 g, 18.6 mmol, 2.5 equiv)
was added in small portions. The mixture was stirred at 85.degree.
C. for 1 h, another 0.73 g zinc powder (11.2 mmol, 1.5 equiv) was
added, and the reaction was stirred at the same temperature
overnight. The reaction was cooled down and filtered, and the
filtrate was concentrated. The residue was then taken into ethyl
acetate and the pH was neutralized with saturated aqueous
NaHCO.sub.3 followed by extraction with ethyl acetate. The organic
layers were combined, dried over anhydrous Na.sub.2SO.sub.4,
concentrated and the remaining residue was purified over a flash
column chromatography yielding 0.96 g (3.2 mmol) of the
intermediate HK-06-205 as a brown solid (43% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 9.16 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.97
(dd, J=1.3 Hz, J=8.0 Hz, 1H), 6.92-6.90 (m, 1H), 5.98 (s, 2H), 4.39
(q, J=7.1 Hz, 2H), 2.36 (s, 3H), 2.22 (s, 3H), 1.44 (t, J=7.1 Hz,
3H).
##STR00300##
Intermediate HK-06-205 (2.92 g, 9.8 mmol) was dissolved in MeCN (30
mL) at room temperature. Dry HCl was bubbled through the mixture
for 30 min and the reaction mixture was refluxed at 85.degree. C.
for 3 h. The reaction was then cooled to room temperature and the
volatile components were removed on a rotary evaporator. The dark
brown solid crude HK-06-208 was used for the next step without
further purification.
##STR00301##
The crude mixture HK-06-208 was dissolved in EtOH (80 mL). 10% NaOH
aqueous solution (40 mL) was added followed by refluxing overnight.
The volatile components were then removed on a rotary evaporator
and the aqueous residue was acidified with 2N HCl aqueous solution.
The brown precipitate was collected by filtration and washed with
water and diethyl ether yielding 2.06 g intermediate HK-06-209 as
brown solid (72% yield over 2 steps).
##STR00302##
Intermediate HK-06-209 (2 g, 6.8 mmol) was mixed with POCl.sub.3
(20 mL) and the mixture was heated at 90.degree. C. for 6 h. The
reaction mixture was cooled to room temperature and the volatile
components were removed on a rotary evaporator. Ethyl acetate (20
mL) was added and the pH was adjusted to 8 with excess saturated
aqueous NaHCO.sub.3 solution. Filtration of the mixture yielded 1.0
g intermediate HK-06-211 as a brown solid in (86% yield). .sup.1H
NMR (DMSO-d.sub.6, 300 MHz): 12.63 (brs, 1H), 8.25 (d, J=8.1 Hz,
1H), 7.54 (d, J=0.8 Hz, 1H), 7.37 (dd, J=8.2 Hz, J=8.1 Hz, 1H),
2.68 (s, 3H), 2.46 (s, 3H), 2.27 (s, 3H).
##STR00303##
To a mixture of HK-06-211 (0.03 g, 0.1 mmol, 1.0 equiv), tert-butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate (0.1 g, 0.3 mmol, 3.0 equiv), and K.sub.2CO.sub.3 (0.07 g,
0.5 mmol, 5.0 equiv), DME (6 mL) and water (4 mL) were added at
room temperature. The mixture was purged with nitrogen before
Pd(PPh.sub.3).sub.4 (0.02 g, 0.02 mmol, 0.02 equiv) was added in
one portion. The reaction mixture was purged with nitrogen and
refluxed at 90.degree. C. for 9 h. The aqueous layer was extracted
with ethyl acetate and combined organic layers were washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, and concentrated. The
residue was taken into 1:1 mixture of CH.sub.2Cl.sub.2 and
trifluoroacetic acid, stirred for 30 min at room temperature. The
mixture was then concentrated and purified with preparative HPLC.
The final compound then dissolved in CH.sub.3CN:H.sub.2O (1:1) and
lyophilized to yield 0.014 g (0.03 mmol) of the final compound Cpd.
No. 114-CF.sub.3CO.sub.2H salt as a bright yellow solid (85%
yield). .sup.1H NMR (CD.sub.3OD, 300 MHz): 7.68-7.66 (m, 1H), 7.56
(d, J=8.7 Hz, 1H), 7.37-7.22 (m, 4H), 7.18-7.12 (m, 1H), 2.97 (s,
3H), 2.60 (s, 3H), 2.45 (s, 3H), 2.28 (s, 3H).
Cpd. No. 115 (TFA salt) was prepared from HK-06-211 using Suzuki
coupling condition [Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 method]
(12% yield)
##STR00304##
.sup.1H NMR (300 MHz, MeOD-d4): 9.32 (d, J=4.60 Hz, 1H), 8.86 (d,
J=8.59 Hz, 1H), 8.09 (d, J=4.63 Hz, 1H), 8.10-8.02 (m, 1H), 7.86
(d, J=8.45 Hz, 1H), 7.77-7.70 (m, 1H), 7.66 (d, J=0.78 Hz, 1H),
7.10 (dd, J=8.24, 1.46 Hz, 1H), 6.91 (d, J=8.22 Hz, 1H), 3.00 (s,
3H), 2.42 (s, 3H), 2.25 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.20N.sub.5O [M+H].sup.+=406.17, Obtained: 406.25
##STR00305## ##STR00306##
A mixture of 2-ethyl-1H-indole (H-11, 0.5 g, 3.4 mmol, 1 equiv) and
N-iodosuccinimide (0.93 g, 4.13 mmol, 1.2 equiv) was stirred in
anhydrous DMF at room temperature overnight. The reaction mixture
was diluted with excess of ethyl acetate, washed with H.sub.2O and
brine. The organic phase was dried over anhydrous Na.sub.2SO.sub.4
and concentrated. The remaining crude HK-06-230 was mixed with
di-tert-butyl dicarbonate (1.5 g, 6.9 mmol) in anhydrous THF. To
this mixture portions of DMAP (0.42 g, 3.44 mmol) was added. The
reaction was stirred at room temperature for 3 h at ambient
atmosphere. Then the mixture was concentrated and the remaining
residue was purified over flash chromatography yielding 0.6 g (1.6
mmol) of intermediate HK-06-231 (47% yield over 2 steps). .sup.1H
NMR (CDCl.sub.3, 300 MHz): 8.09-8.04 (m, 1H), 7.39-7.23 (m, 3H),
3.18 (q, J=7.4 Hz, 2H), 1.69 (s, 9H), 1.23 (t, J=7.4 Hz, 3H).
##STR00307##
To a solution of intermediate HK-06-231 (0.6 g, 1.6 mmol) in
anhydrous THF at -78.degree. C., n-BuLi (2.9 mmol, 1.8 equiv) was
added dropwise. The reaction mixture was stirred at -78.degree. C.
for 30 min. Then to this mixture
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.6 g, 2
equiv) was added and the reaction was stirred for 2 h at
-78.degree. C. The reaction mixture was quenched with aqueous
ammonium chloride solution and extracted to ethyl acetate. The
organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The remaining crude was purified over flash
chromatography yielding 0.39 g (1.04 mmol) of intermediate
HK-06-232 (65% yield). .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.09-7.95
(m, 2H), 7.26-7.17 (m, 2H), 3.37 (q, J=7.3 Hz, 2H), 1.68 (s, 9H),
1.36 (s, 12H), 1.24 (t, J=7.4 Hz, 3H).
##STR00308##
To a mixture of S13 (0.03 g, 0.1 mmol, 1.0 equiv), HK-06-232 (0.11
g, 0.3 mmol, 3.0 equiv) and K.sub.2CO.sub.3 (0.07 g, 0.5 mmol, 5.0
equiv), DME (6 mL) and water (4 mL) were added at room temperature.
The mixture was purged with nitrogen before Pd(PPh.sub.3).sub.4
(0.02 g, 0.02 mmol, 0.02 equiv) was added in one portion. The
reaction mixture was purged with nitrogen and refluxed at
90.degree. C. for overnight. The aqueous layer was extracted with
ethyl acetate and combined organic layers were washed with brine,
dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The residue
was taken into 1:1 mixture of CH.sub.2Cl.sub.2 and trifluoroacetic
acid, stirred for 30 min at room temperature. The mixture was then
concentrated and purified with preparative HPLC. The final compound
then dissolved in CH.sub.3CN:H.sub.2O (1:1) and lyophilized to
yield 0.018 g (0.03 mmol) of the final compound Cpd. No. 116 (TFA
salt) as a bright yellow solid (30% yield). .sup.1H NMR
(CD.sub.3OD, 300 MHz): 7.64-7.59 (m, 1H), 7.53 (s, 1H), 7.35-7.25
(m, 2H), 7.22-7.15 (m, 1H), 6.74 (s, 1H), 3.32 (s, 3H), 3.06 (dq,
J=3.6 Hz, J=7.6 Hz, 2H), 2.96 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H),
1.33 (t, J=7.6 Hz, 3H).
##STR00309## ##STR00310##
4.5 g (23.8 mmol) of 1-bromo-4-fluoro-2-methylbenzene was added to
a mixture of 1.5 ml conc. H.sub.2SO.sub.4 and 1.5 ml fuming nitric
acid at 0.degree. C. and the reaction was stirred at the same
temperature for 1 h. The reaction mixture was poured into ice-water
and extracted to CH.sub.2Cl.sub.2. Combined organic layers were
dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The
remaining residue was purified over flash chromatography yielding
intermediate 1-bromo-4-fluoro-2-methyl-5-nitrobenzene (HK-06-237)
as bright yellow liquid (80% yield). .sup.1H NMR (CDCl.sub.3, 300
MHz): 8.26 (d, J=7.1 Hz, 1H), 7.20 (d, J=11.4 Hz, 1H), 2.48 (s,
3H).
##STR00311##
To a mixture of
3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(0.89 g, 4 mmol, 2 equiv), 1-bromo-4-fluoro-2-methyl-5-nitrobenzene
(0.47 g, 2 mmol, 1 equiv), and K.sub.2CO.sub.3 (0.83 g, 6 mmol, 3.0
equiv), DME (24 mL) and water (16 mL) were added at room
temperature. The mixture was purged with nitrogen before
Pd(PPh.sub.3).sub.4 (0.12 g, 0.1 mmol, 0.05 equiv) was added in one
portion. The reaction mixture was purged with nitrogen and refluxed
at 90.degree. C. overnight. The aqueous layer was extracted with
ethyl acetate and combined organic layers were washed with brine,
dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The residue
was purified over flash chromatography yielding 0.3 g (1.2 mmol) of
the intermediate HK-06-238 as a bright yellow solid (60% yield).
.sup.1H NMR (CDCl.sub.3, 300 MHz): .sup.1H NMR (CDCl.sub.3, 300
MHz): 7.85 (d, J=7.5 Hz, 1H), 7.26 (d, J=11.5 Hz, 1H), 2.27 (s,
3H), 2.23 (s, 3H), 2.11 (s, 3H).
##STR00312##
To a suspension of NaH (0.09 g, 60% in mineral oil, 2.2 mmol, 2.0
equiv) in dry DMF (3 mL), ethyl cyanoacetate (0.19 g, 1.65 mmol,
1.5 equiv) was added dropwise at 0.degree. C. and the reaction was
stirred at room temperature for 30 min. The mixture was cooled to
0.degree. C., anhydrous DMF solution (2 mL) of HK-06-238 (0.28 g,
1.1 mmol, 1.0 equiv) was added via a syringe. The reaction mixture
was allowed to warm up to room temperature and was stirred
overnight. Ethyl acetate (10 ml) and MeOH (5 ml) were added to the
reaction mixture, and pH was adjusted to 2-3 with 2 N HCl aqueous
solution. The volatile components were removed on a rotary
evaporator and the residue was purified over flash chromatography
yielding 0.34 g (1 mmol) of the intermediate HK-06-245 (90% yield).
.sup.1H NMR (CDCl.sub.3, 300 MHz): 8.01 (s, 1H), 7.70 (s, 1H), 5.69
(s, 1H), 4.35 (q, J=7.1 Hz, 2H), 2.34-2.26 (m, 6H), 2.14 (d, J=8.2
Hz, 3H), 1.37 (t, J=7.1 Hz, 3H).
##STR00313##
Acetic acid (6 mL) solution of HK-06-245 (0.32 g, 1.06 mmol, 1.0
equiv) at 80.degree. C., zinc powder (0.17 g, 2.7 mmol, 2.5 equiv)
was added in small portions. The mixture was stirred at 85.degree.
C. for 1 h, another 0.10 g zinc powder (1.6 mmol, 1.5 equiv) was
added, and the reaction was stirred at the same temperature for 3
h. The reaction was cooled down and filtered, thereafter, the
filtrate was concentrated. The residue was then taken into ethyl
acetate and the pH was neutralized with saturated NaHCO.sub.3
followed by extraction with ethyl acetate. The organic layers were
combined, dried over anhydrous Na.sub.2SO.sub.4, concentrated and
the remaining residue was purified over flash chromatography
yielding 0.12 g (0.38 mmol) of the intermediate HK-06-248 (43%
yield).
##STR00314##
Intermediate HK-06-248 (0.8 g, 2.5 mmol) was dissolved in MeCN (20
mL) at room temperature. Dry HCl was bubbled through the mixture
for 30 min and the reaction mixture was refluxed at 85.degree. C.
for 3 h. The reaction was then cooled to room temperature and the
volatile components were removed on a rotary evaporator. The brown
solid crude (HK-06-249) was used for the next step without further
purification.
##STR00315##
The crude mixture HK-06-249 was dissolved in EtOH (20 mL) and 10%
NaOH aqueous solution (10 mL) was added followed by refluxing
overnight. The volatile components were then removed on a rotary
evaporator and the aqueous residue was acidified with 2N HCl
aqueous solution. The brown precipitate was filtered, washed with
water and diethyl ether yielding intermediate HK-06-250 as a bright
brown solid (0.63 g, 81% yield over 2 steps). .sup.1H NMR
(DMSO-d.sub.6, 300 MHz): 12.13 (s, 1H), 11.95 (s, 1H), 7.89 (s,
1H), 7.17 (s, 1H), 2.41 (s, 3H), 2.21 (s, 3H), 2.17 (s, 3H), 2.02
(s, 3H).
##STR00316##
Intermediate HK-06-250 (0.31 g, 1 mmol) was mixed with POCl.sub.3
(3.5 mL) and the mixture was heated at 90.degree. C. for 6.5 h. The
reaction mixture was cooled to room temperature and the volatile
components were removed on a rotary evaporator. Ethyl acetate (5
mL) was added and the pH was adjusted to 8 with excess saturated
aqueous NaHCO.sub.3 solution. Filtration of the mixture yielded
0.33 g intermediate HK-06-255 as brown solid in (99% yield).
.sup.1H NMR (DMSO-d.sub.6, 300 MHz): 12.53 (s, 1H), 8.17 (s, 1H),
7.36 (s, 1H), 2.68 (s, 3H), 2.24 (s, 3H), 2.24 (s, 3H), 2.04 (s,
3H).
##STR00317##
To a mixture of HK-06-255 (0.03 g, 0.1 mmol, 1.0 equiv),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolone (0.05 g,
0.3 mmol, 3.0 equiv), and K.sub.2CO.sub.3 (0.07 g, 0.5 mmol, 5.0
equiv), DME (6 mL) and water (4 mL) were added at room temperature.
The mixture was purged with nitrogen before Pd(PPh.sub.3).sub.4
(0.02 g, 0.02 mmol, 0.02 equiv) was added in one portion. The
reaction mixture was purged with nitrogen and refluxed at
90.degree. C. for overnight. The aqueous layer was extracted with
ethyl acetate and combined organic layers were washed with brine,
dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The residue
was then purified with preparative HPLC. The final compound
dissolved in CH.sub.3CN:H.sub.2O (1:1) and lyophilized to yield
0.01 g (0.02 mmol) of the final compound Cpd. No. 117 (TFA salt) as
a bright yellow solid (17% yield). ESI-MS calculated for
C.sub.26H.sub.22N.sub.5O [M+H].sup.+=420.18, Obtained: 420.42.
##STR00318##
3,5-diethylisoxazole (125 mg) was dissolved in anhydrous AcOH (15
mL). NBS (178 mg) was added and the mixture was heated at reflux
for 2 h before quenching with statured aqueous
Na.sub.2S.sub.2O.sub.3 solution. The aqueous layer was extracted
with ethyl acetate (50 mL.times.3) and the combined organic layers
were washed with brine and dried over anhydrous sodium sulfate. The
solvent was removed on a rotary evaporator and the residue was
purified by flash column chromatography to furnish ZBA18 in 183 mg
(90% yield). ESI-MS calculated for C.sub.7H.sub.10BrNO
[M+H].sup.+=204.00, Obtained: 204.23.
ZBA18 (312 mg) was dissolved in anhydrous THF (15 mL). The solution
was cooled to -78.degree. C. in a dry ice-ethanol bath. BuLi (0.94
mL, 2.5 M in THF) was added dropwise and the mixture was stirred at
-78.degree. C. for 15 min.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (506 mg) was
added via a syringe and the reaction mixture was stirred at
-78.degree. C. for 3 h before quenching with statured aqueous
NH.sub.4Cl solution. The aqueous layer was extracted with ethyl
acetate (50 mL.times.3) and the combined organic layers were washed
with brine and dried over anhydrous sodium sulfate. The solvent was
removed on a rotary evaporator and the residue was purified by
flash column chromatography to furnish 2-trifluoromethylquinoline
4-boronic acid pinacol ester ZBA24 in 310 mg (80% yield).
Cpd. No. 118-TFA salt was prepared from Suzuki coupling of ZBA24
and S13 using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M) condition.
40% yield. .sup.1H NMR (300 MHz, MeOD-d4) .delta. 7.61 (s, 1H),
7.00 (s, 1H), 3.78 (s, 3H), 2.97 (s, 3H), 2.95-2.81 (m, 2H), 2.73
(q, J=7.5 Hz, 2H), 2.34 (s, 3H), 2.16 (s, 3H), 1.27 (t, J=7.6 Hz,
3H), 1.19 (t, J=7.5 Hz, 3H).
##STR00319##
5-Ethyl-3-phenylisoxazole (173 mg) was dissolved in anhydrous AcOH
(15 mL). NBS (178 mg) was added and the mixture was heated at
reflux for 2 h before quenching with statured aqueous
Na.sub.2S.sub.2O.sub.3 solution. The aqueous layer was extracted
with ethyl acetate (50 mL.times.3) and the combined organic layers
were washed with brine and dried over anhydrous sodium sulfate. The
solvent was removed on a rotary evaporator and the residue was
purified by flash column chromatography to furnish ZBA22 in 226 mg
(90% yield). ESI-MS calculated for C11H11BrNO [M+H].sup.+=252.00,
Obtained: 252.43.
ZBA22 (350 mg) was dissolved in anhydrous THF (15 mL). The solution
was cooled to -78.degree. C. in a dry ice-ethanol bath. BuLi (0.94
mL, 2.5 M in THF) was added dropwise and the mixture was stirred at
-78.degree. C. for 15 min.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (506 mg) was
added via a syringe and the reaction mixture was stirred at
-78.degree. C. for 3 h before quenching with statured aqueous
NH.sub.4Cl solution. The aqueous layer was extracted with ethyl
acetate (50 mL.times.3) and the combined organic layers were washed
with brine and dried over anhydrous sodium sulfate. The solvent was
removed on a rotary evaporator and the residue was purified by
flash column chromatography to furnish 2-trifluoromethylquinoline
4-boronic acid pinacol ester ZBA23 in 291 mg (70% yield).
Cpd. No. 119-TFA salt was prepared from Suzuki coupling of ZBA23
and S13 using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M) condition.
40% yield. .sup.1H NMR (300 MHz, MeOD-d4) .delta. 7.71-7.57 (m,
2H), 7.52-7.29 (m, 4H), 6.86 (s, 1H), 3.60 (s, 3H), 2.94 (s, 3H),
2.34 (s, 3H), 2.28 (s, 3H), 2.09 (s, 3H).
##STR00320##
Cpd. No. 120: To a solution of Cpd. No. 73 (43.5 mg) in DMF, NaH (4
mg) and CH.sub.3I (20 mg) were added. The mixture was stirred at
room temperature for 0.5 h. Then water was added and the aqueous
layer extracted with EtOAc. The combined EtOAc extracts were washed
with H.sub.2O, dried over Na.sub.2SO.sub.4, and concentrated under
reduced pressure to afford Cpd. No. 120 (35 mg) after HPLC
purification. .sup.1H NMR (300 MHz, MeOD-d4) .delta. 9.17 (d, J=4.4
Hz, 1H), 8.29 (d, J=8.5 Hz, 1H), 7.91 (ddd, J=8.4, 6.9, 1.4 Hz,
1H), 7.83 (d, J=4.4 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.59 (ddd,
J=8.2, 6.9, 1.1 Hz, 1H), 7.53 (s, 1H), 6.21 (s, 1H), 4.03 (s, 3H),
3.20 (s, 3H), 2.92 (s, 3H), 2.28 (s, 3H), 2.09 (s, 3H).
##STR00321##
Cpd. No. 121: this compound was prepared from Cpd. No. 73 and BnBr
using the same preparation method as Cpd. No. 120. .sup.1H NMR (300
MHz, MeOD-d4) .delta. 9.18 (d, J=4.4 Hz, 1H), 8.29 (d, J=8.5 Hz,
1H), 7.95-7.83 (m, 2H), 7.79 (d, J=8.4 Hz, 1H), 7.60 (ddd, J=8.3,
6.9, 1.2 Hz, 1H), 7.40-7.23 (m, 6H), 6.22 (s, 1H), 5.80 (s, 2H),
3.18 (s, 3H), 2.95 (s, 3H), 2.10 (s, 3H), 1.95 (s, 3H).
##STR00322##
To a solution of S13 (40 mg) and 2-amino-1H-benzimidazole (40 mg)
in DMSO (4 mL), Cs.sub.2CO.sub.3 (60 mg) was added. The mixture was
stirred at 100.degree. C. for 12 h. Then water was added the
aqueous layer extracted with EtOAc. The combined EtOAc extracts
were washed with H.sub.2O, dried over Na.sub.2SO.sub.4, and
concentrated under reduced pressure to afford Cpd. No. 122-TFA salt
(20 mg) after HPLC purification. .sup.1H NMR (300 MHz, MeOD)
.delta. 7.65 (d, J=8.0 Hz, 1H), 7.56-7.41 (m, 2H), 7.32 (t, J=7.8
Hz, 1H), 7.09 (d, J=8.1 Hz, 1H), 6.62 (s, 1H), 3.41 (s, 3H), 2.89
(s, 3H), 2.30 (s, 3H), 2.12 (s, 3H).
##STR00323##
To a solution of S13 (40 mg) and oxindole (40 mg) in THF (6 mL),
K.sub.2CO.sub.3 (60 mg), Pd.sub.2(dba).sub.3 (17 mg), and xPhos (70
mg) were added. The mixture was stirred at 100.degree. C. for 24 h.
Then water was added and the aqueous layer was extracted with
EtOAc. The combined EtOAc extracts were washed with H.sub.2O, dried
over Na.sub.2SO.sub.4, and concentrated under reduced pressure to
afford Cpd. No. 123(4 mg) after HPLC purification. ESI-MS
calculated for C.sub.25H.sub.22N.sub.5O.sub.3 [M+H].sup.+=440.17,
Obtained: 440.32.
##STR00324##
Cpd. No. 124: this compound was prepared from S13 and N-methyl
oxindole using the same method as Cpd. No. 123. ESI-MS calculated
for C.sub.26H.sub.24N.sub.5O.sub.3 [M+H].sup.+=454.18, Obtained:
454.34.
##STR00325##
To a round-bottom flask, S6 (0.37 g, 1.1 mmol) and ethyl
cyanoformate (3 mL) were added at room temperature. Hydrogen
chloride solution in dioxane was added and the reaction mixture was
warmed up to reflux (82.degree. C.) for 2.5 h. The reaction was
then cooled to room temperature and the volatile components were
removed on a rotary evaporator. To this crude mixture, 10% NaOH
aqueous solution (20 mL) and EtOH (50 mL) were added and the
solution was heated at reflux for 6 h. The volatile components were
then removed on a rotary evaporator and the aqueous residue was
acidified with 2N HCl aqueous solution. The product ZBA89 was
allowed to precipitate at 0.degree. C. Filtration of the mixture
furnished pure ZBA89 as a solid in 0.31 g (80% yield, 2 steps).
ESI-MS calculated for C.sub.17H.sub.15N.sub.4O.sub.5
[M+H].sup.+=355.10, Obtained: 355.45.
##STR00326##
To a round-bottom flask, EDCI (0.7 g) and DMAP (0.1 g) were added
to a solution of ZBA89 (0.2 g) in MeOH (100 mL) and DCM (30 mL) at
room temperature. The mixture was stirred for 2 days and the
volatile components were removed on a rotary evaporator. Then ethyl
acetate (40 mL) was added. The product ZBA97 was allowed to
precipitate. Filtration of the mixture furnished pure ZBA97 as a
solid in 0.12 g (60% yield). .sup.1H NMR (300 MHz, MeOD-d4) .delta.
7.86 (s, 1H), 7.39 (s, 1H), 4.07 (s, 3H), 3.93 (s, 3H), 2.34 (s,
3H), 2.17 (s, 3H).
##STR00327##
To a round-bottom flask, ZBA97 (0.278 g) and POCl.sub.3 (8 mL) were
added. The mixture was heated at 90.degree. C. for 6 h. The
reaction mixture was cooled to room temperature and the volatile
components were removed on a rotary evaporator. Water (20 mL) and
ethyl acetate (20 mL) were added and the pH was adjusted to 8 using
NaHCO.sub.3 saturated aqueous solution. Filtration of the mixture
furnished ZBA104 as a brown solid in 0.208 g. .sup.1H NMR (300 MHz,
MeOD-d4) .delta. 8.02 (s, 1H), 7.55 (s, 1H), 4.07 (s, 3H), 3.99 (s,
3H), 2.37 (s, 3H), 2.20 (s, 3H).
##STR00328##
CH.sub.3MgBr solution (0.13 mL, 3 M in Et.sub.2O) was added to a
solution of ZBA104 (40 mg) in THF at room temperature. The mixture
was stirred for 2 h and aq. NH.sub.4Cl solution was added. The
aqueous layer was extracted with EtOAc. The combined EtOAc extracts
were washed with H.sub.2O, dried over Na.sub.2SO.sub.4, and
concentrated under reduced pressure to afford ZBA116 (37 mg).
.sup.1H NMR (300 MHz, MeOD-d4) .delta. 7.93 (s, 1H), 7.45 (s, 1H),
3.96 (s, 3H), 2.36 (s, 3H), 2.19 (s, 3H), 1.67 (s, 6H).
##STR00329##
Cpd. No. 125-TFA salt was prepared from Suzuki coupling of ZBA116
and quinolin-4-ylboronic acid using
Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M) condition. 40% yield.
.sup.1H NMR (300 MHz, MeOD-d4) .delta. 9.33 (d, J=4.8 Hz, 1H), 8.39
(d, J=8.6 Hz, 1H), 8.21-8.03 (m, 2H), 7.94 (d, J=8.4 Hz, 1H),
7.85-7.71 (m, 1H), 7.52 (s, 1H), 6.29 (s, 1H), 3.26 (s, 3H), 2.29
(s, 3H), 2.10 (s, 3H), 1.81 (s, 6H).
##STR00330##
Cpd. No. 126-TFA salt was prepared from Suzuki coupling of ZBA116
and CD223 using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M)
condition. 40% yield. .sup.1H NMR (300 MHz, MeOD) .delta. 9.29 (d,
J=4.6 Hz, 1H), 8.44 (dd, J=9.3, 5.3 Hz, 1H), 8.11 (d, J=4.5 Hz,
1H), 7.91-7.82 (m, 1H), 7.61 (dd, J=9.4, 2.7 Hz, 1H), 7.58 (s, 1H),
6.30 (s, 1H), 3.30 (s, 3H), 2.29 (s, 3H), 2.10 (s, 3H), 1.84 (s,
6H). ESI-MS calculated for C.sub.28H.sub.25FN.sub.5O.sub.3
[M+H].sup.+=498.19, Obtained: 498.54.
##STR00331##
Cpd. No. 127-TFA salt was prepared from Suzuki coupling of ZBA116
and CD224 using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M)
condition. 40% yield. .sup.1H NMR (300 MHz, MeOD) .delta. 9.28 (t,
J=7.9 Hz, 1H), 8.36 (d, J=8.8 Hz, 1H), 8.10 (d, J=4.5 Hz, 1H),
8.02-7.90 (m, 2H), 7.57 (s, 1H), 6.32 (s, 1H), 3.30 (s, 2H), 2.28
(s, 3H), 2.09 (s, 3H), 1.84 (s, 6H). ESI-MS calculated for
C.sub.28H.sub.25ClN.sub.5O.sub.3 [M+H].sup.+=514.16, Obtained:
514.36.
##STR00332##
Azetidine-3-carbonitrile (1.8 g) was dissolved in DCM (50 ml). TsCl
(3.1 g) and Et.sub.3N (6.3 mL) were added and the mixture was
stirred for 3 h. Aq. Brine was added and the aqueous layer was
extracted with DCM. The combined DCM extracts were washed with
H.sub.2O, dried over Na.sub.2SO.sub.4, and concentrated under
reduced pressure to afford ZBA132 (1.9 g) after flash column
chromatography.
To a round-bottom flask, S6 (0.37 g, 1.1 mmol) and ZBA132 (2 g)
were added at room temperature. Hydrogen chloride solution in
dioxane (40 mL) was added and the reaction mixture was warmed up to
reflux (82.degree. C.) for 2.5 h. The reaction was then cooled to
room temperature and the volatile components were removed on a
rotary evaporator. To this crude mixture, 10% NaOH aqueous solution
(20 mL) and EtOH (50 mL) were added and the solution was heated at
reflux for 6 h. The volatile components were then removed on a
rotary evaporator and the aqueous residue was acidified with 2N HCl
aqueous solution. Water was removed on a rotary evaporator and the
product Cpd. No. 128 (40 mg) was obtained after HPLC purification.
ESI-MS calculated for C.sub.26H.sub.26N.sub.5O.sub.5S
[M+H].sup.+=520.16, Obtained: 520.55.
##STR00333##
To a round-bottom flask, ZBA104 (0.038 g, 0.1 mmol) was dissolved
in THF (7 mL) at room temperature. LiAlH.sub.4 (7.6 mg, 0.2 mmol)
was added and the reaction mixture was stirred for 2.5 h. Then
water and Ethyl acetate was slowly added. The aqueous layer was
extracted with EtOAc. The combined EtOAc extracts were washed with
H.sub.2O, dried over Na.sub.2SO.sub.4, and concentrated under
reduced pressure to afford ZBA139 (27 mg). ESI-MS calculated for
C.sub.17H.sub.15ClN.sub.4O.sub.3 [M+H].sup.+=359.09, Obtained:
359.43.
Cpd. No. 129-TFA salt was prepared from Suzuki coupling of ZBA139
and quinolin-4-ylboronic acid using
Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M) condition. 38% yield.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.49 (d, J=5.0 Hz, 1H), 8.50
(d, J=8.5 Hz, 1H), 8.36 (d, J=5.0 Hz, 1H), 8.16 (t, J=7.7 Hz, 1H),
8.07 (d, J=8.4 Hz, 1H), 7.85 (t, J=7.6 Hz, 1H), 7.58 (s, 1H), 6.36
(s, 1H), 5.13 (s, 2H), 3.30 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H).
ESI-MS calculated for C.sub.26H.sub.22N.sub.5O.sub.3
[M+H].sup.+=452.17, Obtained: 452.57.
##STR00334##
To a round-bottom flask, Cpd. No. 129 (0.045 g, 0.1 mmol) was
dissolved in DCM (7 mL) and Pyridine (0.4 mL) at room temperature.
Dess-martin periodinane (63.6 mg, 0.15 mmol) was added and the
reaction mixture was stirred for 2.5 h. Then water and ethyl
acetate was slowly added. The aqueous layer was extracted with
EtOAc. The combined EtOAc extracts were washed with H.sub.2O, dried
over Na.sub.2SO.sub.4, and concentrated under reduced pressure to
afford the aldehyde intermediate ZBA154. The intermediate ZBA154,
morpholine (0.3 mL) and NaBH(OAc).sub.3 (90 mg, 0.4 mmol) was
dissolved in ClCH.sub.2CH.sub.2Cl (10 mL) and the mixture was
stirred overnight. Then water and Ethyl acetate was slowly added.
The aqueous layer was extracted with EtOAc. The combined EtOAc
extracts were washed with H.sub.2O, dried over Na.sub.2SO.sub.4,
and concentrated under reduced pressure to afford Cpd. No. 130 (24
mg) after HPLC purification. .sup.1H NMR (300 MHz, MeOD) .delta.
9.33 (d, J=4.9 Hz, 1H), 8.39 (d, J=8.5 Hz, 1H), 8.13 (d, J=4.9 Hz,
1H), 8.08 (ddd, J=8.5, 6.9, 1.3 Hz, 1H), 8.00-7.93 (m, 1H), 7.75
(ddd, J=8.3, 6.9, 1.1 Hz, 1H), 7.49 (s, 1H), 6.37 (s, 1H), 4.87 (s,
2H), 4.04 (brs, 4H), 3.66 (brs, 4H), 3.27 (s, 3H), 2.28 (s, 3H),
2.09 (s, 3H). ESI-MS calculated for C.sub.30H.sub.29N.sub.6O.sub.3
[M+H].sup.+=521.23, Obtained: 521.67.
##STR00335##
Cpd. No. 131-TFA salt was prepared from reductive amination of
ZBA154 and dimethylamine using NaBH(OAc).sub.3 condition. 50%
yield. .sup.1H NMR (300 MHz, MeOD) .delta. 9.40 (d, J=5.0 Hz, 1H),
8.44 (d, J=8.5 Hz, 1H), 8.24 (d, J=5.0 Hz, 1H), 8.19-8.10 (m, 1H),
8.04 (d, J=8.3 Hz, 1H), 7.81 (t, J=7.4 Hz, 1H), 7.50 (s, 1H), 6.40
(s, 1H), 4.83 (s, 2H), 3.28 (s, 3H), 3.16 (s, 6H), 2.28 (s, 3H),
2.09 (s, 3H). ESI-MS calculated for C.sub.28H.sub.27N.sub.6O.sub.2
[M+H].sup.+=479.21, Obtained: 479.44.
##STR00336##
Cpd. No. 132-TFA salt was prepared from reductive amination of
ZBA154 and 1-methylpiperazine using NaBH(OAc).sub.3 condition. 50%
yield. .sup.1H NMR (300 MHz, MeOD) .delta. 9.32 (d, J=4.8 Hz, 1H),
8.38 (d, J=8.6 Hz, 1H), 8.16-8.01 (m, 2H), 7.93 (d, J=8.2 Hz, 1H),
7.81-7.68 (m, 1H), 7.49 (s, 1H), 6.32 (s, 1H), 4.44 (s, 2H),
3.58-3.32 (m, 8H), 3.26 (s, 3H), 2.95 (s, 3H), 2.28 (s, 3H), 2.10
(s, 3H). ESI-MS calculated for C.sub.31H.sub.32N.sub.7O.sub.2
[M+H].sup.+=534.26, Obtained: 534.55.
##STR00337##
Cpd. No. 133-TFA salt was prepared from reductive amination of
ZBA154 and diethylamine using NaBH(OAc).sub.3 condition. 50% yield.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.35 (d, J=4.9 Hz, 1H), 8.40
(d, J=8.5 Hz, 1H), 8.15 (d, J=4.9 Hz, 1H), 8.09 (ddd, J=8.5, 6.9,
1.3 Hz, 1H), 7.99 (d, J=7.9 Hz, 1H), 7.76 (ddd, J=8.3, 6.9, 1.1 Hz,
1H), 7.50 (s, 1H), 6.39 (s, 1H), 4.83 (s, 2H), 3.65-3.40 (m, 4H),
3.28 (s, 3H), 2.28 (s, 3H), 2.09 (s, 3H), 1.47 (t, J=7.2 Hz, 6H).
ESI-MS calculated for C.sub.30H.sub.31N.sub.6O.sub.2
[M+H].sup.+=507.25, Obtained: 507.44.
##STR00338##
Cpd. No. 134-TFA salt was prepared from reductive amination of
ZBA154 and pyrrolidine using NaBH(OAc).sub.3 condition. 53% yield.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.31 (d, J=4.8 Hz, 1H), 8.38
(d, J=8.5 Hz, 1H), 8.14-8.01 (m, 2H), 7.94 (d, J=7.9 Hz, 1H),
7.77-7.70 (m, 1H), 7.48 (s, 1H), 6.35 (s, 1H), 4.91 (s, 2H),
4.05-3.85 (m, 2H), 3.51-3.31 (m, 2H), 3.26 (s, 3H), 2.39-2.00 (m,
10H). ESI-MS calculated for C.sub.30H.sub.29N.sub.6O.sub.2
[M+H].sup.+=505.23, Obtained: 505.43.
The same reaction conditions for the synthesis of S13 can be used
to synthesize S16 (scheme below). Reflux S6 with isobutyronitrile
in the presence of dry HCl will afford compound 14, which will
readily cyclized into S15 upon treatment of base (NaOH-water-EtOH)
at 120.degree. C. Treatment of S15 with POCl.sub.3 will afford S16,
a key intermediate can undergo direct condensation/coupling
reaction with amine and Suzuki coupling reaction with aromatic
pinacol boronate or vinyl pinacol boronate. An example of Suzuki
coupling of S16 and tert-Butyl
2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carb-
oxylate is depicted in the following scheme to give Cpd. No.
135.
##STR00339##
The same reaction conditions for the synthesis of S13 can be used
to synthesize S20 (scheme below). A similar method to synthesis of
4-chloro-9H-pyrimido[4,5-b]indol-2-amine from ethyl
2-amino-1H-indole-3-carboxylate has been reported by H. D. Hollisin
Showalter and cowrkers in Journal of Medicinal Chemistry (J. Med.
Chem. 1999, 42, 5464-5474). Reflux S6 with cyanamide in the
presence of concentrated HCl in 1,4-dioxane will afford
intermediate S18, which will readily cyclized into S19 upon
treatment of base (NaOH-water-EtOH) at relux. Treatment of S19 with
POCl.sub.3 at 90.degree. C. will afford S20.
S20 is a key intermediate that can undergo direct
condensation/coupling reaction with amine and Suzuki coupling
reaction with aromatic pinacol boronate or vinyl pinacol boronate.
An example of Suzuki coupling of S20 and commercially available
quinolin-4-ylboronic acid was depicted in the following scheme.
##STR00340##
Standard reductive amine of S21 and a variety of aldehydes in the
presence of NaBH(OAc).sub.3 and acetic acid in 1,2-dichloroethane
will give the corresponding product S22.
##STR00341##
4-Bromo-2-(4-methylpiperazin-1-yl)quinoline (CE46)
2,4-Dibromoquinoline (572 mg, 2.0 mmol) and 1-methyl-piperazine
(200 mg, 2.0 mmol) were dissolved in anhydrous DMSO (6 mL). The
solution was heated at 90.degree. C. for 16 h. The reaction was
quenched with water. The aqueous layer was extracted with ethyl
acetate and the combined organic layers were washed with brine, dry
over anhydrous sodium sulfate, and concentrated on a rotary
evaporator. The residue was purified by flash column chromatography
to yield 4-bromo-2-(4-methylpiperazin-1-yl)quinoline and its region
isomer 2-bromo-4-(4-methylpiperazin-1-yl)quinoline in 0.50 g (ratio
2:1). The mixture of two isomers was used for synthesis of CE52
without further purification. .sup.1H NMR (CDCl.sub.3, 300 MHz):
7.94 (d, J=8.19 Hz, 1H), 7.65 (d, J=8.14 Hz, 1H), 7.62-7.54 (m,
1H), 7.33 (ddd, J=8.11, 6.79, 1.16 Hz, 1H), 7.26 (s, 1H), 4.10-3.90
(m, 4H), 3.30-3.05 (m, 4H), 2.57 (s, 3H).
2-(4-Methylpiperazin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
quinoline (CE52)
4-Bromo-2-(4-methylpiperazin-1-yl)quinoline and its region isomer
(0.50 g, 1.6 mmol., 1.0 equiv.), bis(pinacolato)diboron (812 mg,
3.2 mmol, 2.0 equiv.), and potassium acetate (640 mg, 6.4 mmol, 4.0
equiv.) were added to a round-bottom flask Anhydrous 1,4-dioxane
(10 mL) was added and the system was degassed and refilled
nitrogen. Pd(dppf)Cl.sub.2 (112 mg, 0.16 mmol, 0.1 equiv.) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to room
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound was isolated in 130 mg
(mixture of isomers). ESI-MS calculated for
C.sub.20H.sub.29BN.sub.3O.sub.2 [M+H].sup.+=354.24; Observed:
354.58.
##STR00342##
4-(6-Methoxy-2-methyl-4-(2-(4-methylpiperazin-1-yl)quinolin-4-yl)-9H-pyrim-
ido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
Suzuki coupling of
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 45 mg) and
2-(4-methylpiperazin-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl-
)quinoline (CE52, 130 mg) using condition Method 42 afforded the
title compound as a salt of CF.sub.3CO.sub.2H (26 mg, 34% yield).
.sup.1H NMR (MeOD-d4, 300 MHz): 8.00 (d, J=8.39 Hz, 1H), 7.81 (s,
1H), 7.85-7.75 (m, 1H), 7.60-7.54 (m, 1H), 7.54 (s, 1H), 7.35 (t,
J=7.27 Hz, 1H), 6.26 (s, 1H), 3.80-3.30 (m, 8H), 3.20 (s, 3H), 3.02
(s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.31H.sub.32N.sub.7O.sub.2 [M+H].sup.+=534.26;
Observed: 534.42.
##STR00343##
N.sup.1-(4-bromoquinolin-2-yl)-N.sup.2,N.sup.2-diethylethane-1,2-diamine
(CE49)
2,4-Dibromoquinoline (861 mg, 3.0 mmol) and
N.sup.1,N.sup.1-diethylethane-1,2-diamine (348 mg, 3.0 mmol) were
dissolved in anhydrous DMSO (6 mL). The solution was heated at
90.degree. C. for 16 h. The reaction was quenched with water. The
pH value of the reaction mixture was adjusted be less than 1 using
CF.sub.3CO.sub.2H and the mixture was purified on reverse phase
HPLC to yield
N.sup.1-(4-bromoquinolin-2-yl)-N.sup.2,N.sup.2-diethylethane-1,2-diamine
as a salt of TFA in 0.30 g (33% yield). The ratio for two region
isomer is ca. 1:1 determined by analytical UPLC. .sup.1H NMR
(MeOD-d4, 300 MHz): 8.08 (d, J=8.20 Hz, 1H), 8.00-7.80 (m, 1H),
7.82 (t, J=7.58 Hz, 1H), 7.57 (t, J=8.12 Hz, 2H), 4.06 (t, J=6.35
Hz, 2H), 3.56 (t, J=6.35 Hz, 2H), 3.35 (q, J=7.39 Hz, 4H), 1.36 (t,
J=7.39 Hz, 6H). ESI-MS calculated for
C.sub.15H.sub.21.sup.79BrN.sub.3 [M+H].sup.+=322.09; Observed:
322.58.
(2-((2-(Diethylamino)ethyl)amino)quinolin-4-yl)boronic acid
(CE55)
N.sup.1-(4-bromoquinolin-2-yl)-N.sup.2,N.sup.2-diethylethane-1,2-diamine
(0.30 g, 1.0 mmol., 1.0 equiv.), bis(pinacolato)diboron (500 mg,
2.0 mmol, 2.0 equiv.), and potassium acetate (400 mg, 4 mmol, 4.0
equiv.) were added to a round-bottom flask Anhydrous 1,4-dioxane
(10 mL) was added and the system was degassed and refilled with
nitrogen. Pd(dppf)Cl.sub.2 (70 mg, 0.1 mmol, 0.1 equiv.) was added
and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to room
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography using MeOH--NH.sub.3 as eluent. The title
compound was obtained in 140 mg (38% yield). .sup.1H NMR (MeOD-d4,
300 MHz): 7.98 (d, J=8.04 Hz, 1H), 7.74 (ddd, J=8.43, 7.20, 1.26
Hz, 1H), 7.96-7.84 (m, 1H), 7.50 (ddd, J=8.43, 7.31, 1.10 Hz, 1H),
7.30-7.20 (m, 1H), 4.08 (t, J=6.57 Hz, 2H), 3.56 (t, J=6.57 Hz,
2H), 3.35 (q, J=7.29, 4H), 1.36 (t, J=7.29 Hz, 6H).
##STR00344##
N.sup.1-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4-
,5-b]indol-4-yl)quinolin-2-yl)-N.sup.2,N.sup.2-diethylethane-1,2-diamine
Suzuki coupling of S13 (70 mg) and
(2-((2-(diethylamino)ethyl)amino)quinolin-4-yl)boronic acid (CE55,
130 mg) using condition Method 42 afforded the title compound (50
mg, 38% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.09 (d, J=8.41 Hz,
1H), 7.88 (t, J=7.75 Hz, 1H), 7.63 (d, J=8.05 Hz, 2H), 7.52 (s,
1H), 7.43 (t, J=7.67 Hz, 1H), 6.49 (s, 1H), 4.15 (t, J=6.29 Hz,
2H), 3.62 (t, J=6.41 Hz, 2H), 3.40 (q, J=7.20 Hz, 1H), 3.31 (s,
3H), 2.99 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H), 1.39 (t, J=7.3 Hz,
6H). ESI-MS calculated for C.sub.32H.sub.36N.sub.7O.sub.2
[M+H].sup.+=550.29; Observed: 550.25.
##STR00345##
4-Bromo-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-2-amine (CE62)
2,4-Dibromoquinoline (861 mg, 3.0 mmol),
2-(pyrrolidin-1-yl)ethanamine (342 mg, 3.0 mmol), and
K.sub.2CO.sub.3 (414 mg, 3.0 mmol) were mixed in anhydrous DMF (6
mL). The solution was heated at 90.degree. C. for 16 h. The pH
value of the reaction mixture was adjusted be less than 1 using
CF.sub.3CO.sub.2H and the mixture was purified on reverse phase
HPLC to yield 4-bromo-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-2-amine
as a salt of TFA in 0.48 g (37% yield).
The ratio for two region isomer is ca. 1:1 determined by analytical
UPLC. .sup.1H NMR (MeOD-d4, 300 MHz): 7.96 (d, J=8.22 Hz, 1H),
7.80-7.70 (m, 2H), 7.60-7.45 (m, 2H), 4.05 (t, J=Hz, 2H), 3.90-3.60
(m, 2H), 3.63 (t, J=6.08 Hz, 2H), 3.30-3.10 (m, 2H), 2.30-2.00 (m,
4H). ESI-MS calculated for C.sub.15H.sub.19.sup.79BrN.sub.3
[M+H].sup.+=320.08; Observed: 320.36.
N-(2-(Pyrrolidin-1-yl)ethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-y-
l)quinolin-2-amine (CE66)
4-Bromo-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-2-amine (0.48 g, 1.11
mmol), bis(pinacolato)-diboron (762 mg, 3.0 mmol), and potassium
acetate (600 mg, 6 mmol) were added to a round-bottom flask
Anhydrous 1,4-dioxane (10 mL) was added and the system was degassed
and refilled with nitrogen. Pd(dppf)Cl.sub.2 (0.105 mg, 0.15 mmol)
was added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to room
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified on reverse
phase HPLC to yield the title compound as a salt of TFA in 320 mg
(44% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.50 (d, J=8.12 Hz,
1H), 8.00-7.80 (m, 1H), 7.72 (t, J=7.63 Hz, 1H), 7.48 (t, J=8.07
Hz, 1H), 7.47 (s, 1H), 4.09 (t, J=5.86 Hz, 2H), 3.64 (t, J=6.03 Hz,
2H), 3.90-3.65 (m, 2H), 3.30-3.10 (m, 2H), 2.30-2.00 (m, 4H), 1.42
(s, 12H). ESI-MS calculated for C.sub.21H.sub.31BN.sub.3O.sub.2
[M+H].sup.+=368.25; Observed: 368.33.
##STR00346##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-(pyrrolidin-1-yl)ethyl)quinolin-2-amine
Suzuki coupling of S13 (136 mg, 0.4 mmol) and
N-(2-(pyrrolidin-1-yl)ethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2--
yl)quinolin-2-amine (CE66, 320 mg, 0.66 mmol) using condition
Method 42 afforded the title compound as a salt of
CF.sub.3CO.sub.2H (80 mg, 30% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.13 (d, J=8.38 Hz, 1H), 7.92 (t, J=7.25 Hz, 1H), 7.67 (d,
J=7.39 Hz, 2H), 7.54 (s, 1H), 7.47 (t, J=7.69 Hz, 1H), 6.52 (s,
1H), 4.16 (t, J=6.11 Hz, 2H), 3.71 (t, J=6.22 Hz, 2H), 3.80-3.60
(m, 2H), 3.40-3.20 (m, 2H), 3.01 (s, 3H), 2.29 (s, 3H), 2.30-2.10
(m, 4H), 2.10 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.34N.sub.7O.sub.2 [M+H].sup.+=548.28: Observed:
548.88.
##STR00347##
4-Bromo-N-(2-morpholinoethyl)quinolin-2-amine (CE60)
2,4-Dibromoquinoline (861 mg, 3.0 mmol), 2-morpholineethanamine
(390 mg, 3.0 mmol), and K.sub.2CO.sub.3 (414 mg, 3.0 mmol) were
mixed in anhydrous DMF (6 mL). The solution was heated at
90.degree. C. for 16 h. The pH value of the reaction mixture was
adjusted be less than 1 using CF.sub.3CO.sub.2H and the mixture was
purified on reverse phase HPLC to yield
4-bromo-N-(2-morpholinoethyl)quinolin-2-amine as a salt of TFA in
0.545 g (1.2 mmol, 40% yield). The ratio for two region isomer is
ca. 1:1 determined by analytical UPLC. .sup.1H NMR (MeOD-d4, 300
MHz): 8.06 (d, J=8.22 Hz, 1H), 7.90-7.70 (m, 2H), 7.70-7.50 (m,
2H), 4.09 (t, J=6.17 Hz, 2H), 4.00-3.85 (m, 4H), 3.60 (t, J=6.17
Hz, 2H), 3.50-3.30 (m, 4H). ESI-MS calculated for
C.sub.15H.sub.19.sup.79BrN.sub.3O [M+H].sup.+=336.07; Observed:
336.16.
N-(2-Morpholinoethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quino-
lin-2-amine (CE71)
4-Bromo-N-(2-morpholinoethyl)quinolin-2-amine (0.54 g, 1.2 mmol),
bis(pinacolato)diboron (838 mg, 3.3 mmol), and potassium acetate
(640 mg, 6.4 mmol) were added to a round-bottom flask Anhydrous
1,4-dixoane (10 mL) was added and the system was degassed and
refilled with nitrogen. Pd(dppf)Cl.sub.2 (112 mg, 0.16 mmol) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to room
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified on reverse
phase flash column chromatography to yield the title compound as a
salt of TFA in 460 mg (77% yield). .sup.1H NMR (MeOD-d4, 300 MHz):
8.49 (d, J=8.11 Hz, 1H), 7.91 (d, J=7.73 Hz, 1H), 7.71 (t, J=7.76
Hz, 1H), 7.48 (t, J=7.53 Hz, 1H), 7.45 (s, 1H), 4.14 (t, J=5.91 Hz,
2H), 4.00-3.80 (m, 4H), 3.61 (t, J=5.91 Hz, 2H), 3.50-3.30 (m, 4H),
1.42 (s, 12H). ESI-MS calculated for
C.sub.21H.sub.31BN.sub.3O.sub.3 [M+H].sup.+=384.25; Observed:
384.50.
##STR00348##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-morpholinoethyl)quinolin-2-amine
Suzuki coupling of S13 (205 mg, 0.6 mmol) and
N-(2-morpholinoethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quin-
olin-2-amine (CE71, 0.46 g, 0.93 mmol) using condition Method 42
afforded the title compound as a salt of CF.sub.3CO.sub.2H (50 mg,
38% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.15 (d, J=7.91 Hz,
1H), 7.91 (t, J=7.79 Hz, 1H), 7.85-7.70 (m, 1H), 7.67 (d, J=7.85
Hz, 1H), 7.53 (s, 1H), 7.54 (t, J=7.71 Hz, 1H), 6.53 (s, 1H),
4.30-4.15 (m, 2H), 4.10-3.90 (m, 4H), 3.75-3.60 (m, 2H), 3.60-3.40
(m, 4H), 3.00 (s, 3H), 2.24 (s, 3H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.32H.sub.34N.sub.7O.sub.3 [M+H].sup.+=564.27;
Observed: 564.67.
##STR00349##
4-(3-((4-Bromoquinolin-2-yl)oxy)propyl)morpholine (CE90)
NaH (80 mg, 60% in mineral oil, 2.0 mmol) and anhydrous DMF (6 mL)
were added to a round-bottom flask. To this flask,
3-morpholinopropan-1-ol (300 mg, 2.0 mmol) was added via a syringe
and the mixture was stirred at room temperature for 20 min.
2,4-Dibromoquinoline (574 mg, 2.0 mmol) was added in one portion
and the mixture was heated at 70.degree. C. for 16 h. The reaction
was quenched with water. The aqueous layer was extracted with ethyl
acetate and the combined organic layers were washed with brine, dry
over anhydrous sodium sulfate, and concentrated on a rotary
evaporator. The residue was purified by flash column chromatography
to yield 4-(3-((4-bromoquinolin-2-yl)oxy)propyl)morpholine in 0.168
g (0.48 mmol, 24% yield). The ratio for two region isomer is ca.
1:1 determined by analytical UPLC. .sup.1H NMR (CDCl.sub.3, 300
MHz): 8.11 (d, J=8.26 Hz, 1H), 7.84 (d, J=8.36 Hz, 1H), 7.69 (ddd,
J=8.36, 7.11, 1.37 Hz, 1H), 7.49 (ddd, J=8.21, 6.86, 1.20 Hz, 1H),
7.28 (s, 1H), 4.56 (t, J=6.46 Hz, 2H), 3.84-3.72 (m, 4H), 2.58 (t,
J=7.11 Hz, 2H), 2.58-2.50n (m, 4H), 2.12-2.02 (m, 2H).
4-(3-((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-2-yl)oxy)pr-
opyl)morpholine (CE95)
4-(3-((4-Bromoquinolin-2-yl)oxy)propyl)morpholine in 0.168 g (0.48
mmol, 1.0 equiv.), bis(pinacolato)diboron (254 mg, 1.0 mmol, 2.0
equiv.), and potassium acetate (200 mg, 2.0 mmol, 4.0 equiv.) were
added to a round-bottom flask Anhydrous 1,4-dixoane (10 mL) was
added and the system was degassed and refilled with nitrogen.
Pd(dppf)Cl.sub.2 (40 mg, 0.05 mmol, 0.1 equiv.) was added and the
system was degassed again followed by heating at 100.degree. C. for
16 h. The reaction mixture was cooled to room temperature and
diluted by CH.sub.2Cl.sub.2. The solution was filtered through a
pad of celite and the volatile components were removed on a rotary
evaporator. The residue was purified by flash column chromatography
to yield the title compound in 80 mg (42% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 8.53 (dd, J=8.23, 1.02 Hz, 1H), 7.85 (dd,
J=8.36, 0.70 Hz, 1H), 7.63 (ddd, J=8.36, 6.97, 1.44 Hz, 1H), 7.43
(ddd, J=8.20, 6.95, 1.29 Hz, 1H), 7.42 (s, 1H), 4.55 (t, J=6.32 Hz,
2H), 3.83-3.76 (m, 4H), 2.63 (t, J=7.36 Hz, 2H), 2.61-2.54 (m, 4H),
2.18-2.00 (m, 2H), 1.45 (s, 12H). ESI-MS calculated for
C.sub.22H.sub.32BN.sub.2O.sub.4 [M+H].sup.+=399.25; Observed:
399.50.
##STR00350##
4-(3-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-
-b]indol-4-yl)quinolin-2-yl)oxy)propyl)morpholine
Suzuki coupling of S13 (40 mg, 0.1 mmol) and
4-(3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-2-yl)oxy)p-
ropyl)morpholine (CE95, 80 mg) using condition Method 42 afforded
the title compound as a salt of CF.sub.3CO.sub.2H (20 mg, 29%
yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.12 (d, J=8.46 Hz, 1H),
7.94-7.84 (m, 1H), 7.68 (d, J=8.31 Hz, 1H), 7.57 (s, 1H), 7.52 (s,
1H), 7.53-7.46 (m, 1H), 6.29 (s, 1H), 4.84-4.70 (m, 2H), 4.20-4.00
(m, 2H), 4.00-3.80 (m, 2H), 3.70-3.50 (m, 2H), 3.55-3.45 (m, 2H),
3.30-3.10 (m, 2H), 3.24 (s, 3H), 3.04 (s, 3H), 2.50-2.36 (m, 2H),
2.31 (s, 3H), 2.12 (s, 3H). ESI-MS calculated for
C.sub.33H.sub.35N.sub.6O.sub.4 [M+H].sup.+=579.27; Observed:
579.33.
##STR00351##
4-Bromo-N-(3-(4-methylpiperazin-1-yl)-propyl)quinolin-2-amine
(CE86)
2,4-Dibromoquinoline (861 mg, 3.0 mmol),
3-(4-methylpiperazin-1-yl)-propan-1-amine (471 mg, 3.0 mmol), and
Na.sub.2CO.sub.3 (315 mg, 3.0 mmol) were mixed in anhydrous DMF (6
mL). The solution was heated at 90.degree. C. for 16 h. The pH
value of the reaction mixture was adjusted be less than 1 using
CF.sub.3CO.sub.2H and the mixture was purified on reverse phase
HPLC to yield
4-bromo-N-(3-(4-methylpiperazin-1-yl)-propyl)quinolin-2-amine as a
salt of TFA in 0.51 g (1.07 mmol, 36% yield). The ratio for two
region isomer is ca. 1:1 determined by analytical UPLC. .sup.1H NMR
(MeOD-d4, 300 MHz): 8.00 (d, J=8.17 Hz, 1H), 7.96-7.82 (m, 1H),
7.77 (ddd, J=8.36, 7.16, 1.15 Hz, 1H), 7.52 (t, J=7.71 Hz, 1H),
7.50-7.40 (m, 1H), 3.70-3.60 (m, 10H), 3.50-3.30 (m, 2H), 3.00 (s,
3H), 2.36-2.16 (m, 2H). ESI-MS calculated for
C.sub.17H.sub.24.sup.79BrN.sub.4 [M+H].sup.+=363.12; Observed:
363.56.
N-(3-(4-Methylpiperazin-1-yl)propyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)quinolin-2-amine (CE98)
4-Bromo-N-(3-(4-methylpiperazin-1-yl)-propyl)quinolin-2-amine (0.51
g, 1.07 mmol), bis(pinacolato)diboron (711 mg, 2.8 mmol), and
potassium acetate (560 mg, 5.6 mmol) were added to a round-bottom
flask Anhydrous 1,4-dixoane (10 mL) was added and the system was
degassed and refilled with nitrogen. Pd(dppf)Cl.sub.2 (98 mg, 0.14
mmol) was added and the system was degassed again followed by
heating at 100.degree. C. for 16 h. The reaction mixture was cooled
to room temperature and diluted by CH.sub.2Cl.sub.2. The solution
was filtered through a pad of celite and the volatile components
were removed on a rotary evaporator. The residue was purified on
reverse phase HPLC to yield the title compound in 600 mg (>90%
yield, with impurity). .sup.1H NMR (MeOD-d4, 300 MHz): 8.54 (d,
J=8.00 Hz, 1H), 8.00-7.80 (m, 1H), 7.75 (t, J=7.73 Hz, 1H),
7.57-7.46 (m, 1H), 7.51 (s, 1H), 3.86-3.66 (m, 10H), 3.52-3.42 (m,
2H), 3.04 (s, 3H), 2.38-2.22 (m, 2H), 1.46 (s, 12H). ESI-MS
calculated for C.sub.23H.sub.36BN.sub.4O.sub.2 [M+H].sup.+=411.29;
Observed: 411.50.
##STR00352##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)quinolin-2-amine
Suzuki coupling of S13 (205 mg, 0.6 mmol) and
N-(3-(4-methylpiperazin-1-yl)propyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxabo-
rolan-2-yl)quinolin-2-amine (CE98, 600 mg) using condition Method
42 afforded the title compound as a salt of CF.sub.3CO.sub.2H (156
mg, 37% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.30-8.10 (m, 1H),
7.92 (t, J=7.66 Hz, 1H), 7.67 (d, J=8.15 Hz, 1H), 7.75-7.60 (m,
1H), 7.55 (s, 1H), 7.47 (t, J=7.76 Hz, 1H), 6.55 (s, 1H), 4.00-3.80
(m, 2H), 3.80-3.60 (m, 8H), 3.55-3.40 (m, 2H), 3.31 (s, 3H), 2.97
(s, 3H), 2.96 (s, 3H), 2.46-2.28 (m, 2H), 1.76 (s, 3H). ESI-MS
calculated for C.sub.34H.sub.39N.sub.8O.sub.2 [M+H].sup.+=591.32;
Observed: 591.50.
##STR00353##
Methyl 3-((4-bromoquinolin-2-yl)amino)propanoate (CE101)
2,4-Dibromoquinoline (861 mg, 3.0 mmol), .beta.-alanine methyl
ester HCl salt (462 mg, 3.3 mmol), and K.sub.2CO.sub.3 (515 mg, 3.7
mmol) were mixed in anhydrous DMSO (6 mL). The solution was heated
at 90.degree. C. for 16 h. The reaction was quenched with water.
The aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine, dry over anhydrous sodium
sulfate, and concentrated on a rotary evaporator. The residue was
purified on flash column chromatography to yield methyl
3-((4-bromoquinolin-2-yl)amino)propanoate in 0.32 g (1.0 mmol, 33%
yield). The ratio for two region isomers is ca. 1:1 determined by
analytical UPLC. .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.93 (dd,
J=8.25, 0.95 Hz, 1H), 7.66 (dd, J=8.39, 0.69 Hz, 1H), 7.54 (ddd,
J=8.36, 6.92, 1.41, 1H), 7.27 (ddd, J=8.18, 6.90, 1.25 Hz, 1H),
6.93 (s, 1H), 5.23 (t, J=5.44 Hz, 1H), 3.81 (q, J=6.12 Hz, 2H),
3.69 (s, 3H), 2.72 (t, J=6.07 Hz, 2H). ESI-MS calculated for
C.sub.13H.sub.14.sup.79BrN.sub.2O.sub.2 [M+H].sup.+=309.02;
Observed: 309.42.
Methyl
3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-2-yl)am-
ino)propanoate (CE108)
Methyl 3-((4-bromoquinolin-2-yl)amino)propanoate (0.42 g, 1.4
mmol), bis(pinacolato)diboron (711 mg, 2.8 mmol), and potassium
acetate (560 mg, 5.6 mmol) were added to a round-bottom flask
Anhydrous 1,4-dixoane (10 mL) was added and the system was degassed
and refilled with nitrogen. Pd(dppf)Cl.sub.2 (98 mg, 0.14 mmol) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to room
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified on reverse
phase flash column chromatography to yield the title compound in
620 mg (with impurity). .sup.1H NMR (MeOD-d4, 300 MHz): 8.34 (d,
J=8.09 Hz, 1H), 7.85-7.70 (m, 1H), 7.70-7.55 (m, 1H), 7.45-7.30 (m,
2H), 3.90-3.70 (m, 2H), 3.68 (s, 3H), 2.90-2.70 (m, 2H), 1.39 (s,
12H). ESI-MS calculated for C.sub.19H.sub.26BN.sub.2O.sub.4
[M+H].sup.+=357.20; Observed: 357.75.
##STR00354##
3-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)-quinolin-2-yl)amino)propanoic acid
Suzuki coupling of S13 (180 mg, 0.5 mmol) and methyl
3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-2-yl)amino)pr-
opanoate (CE108, 620 mg, 1.32 mmol) using condition Method 42
afforded the title compound in 30 mg (9% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.11 (d, J=7.02 Hz, 1H), 7.90 (td, J=7.87, 1.00
Hz, 1H), 7.65 (d, J=7.78 Hz, 1H), 7.49 (s, 1H), 7.50-7.42 (m, 2H),
6.65 (s, 1H), 4.10-3.86 (m, 2H), 3.40 (s, 3H), 2.94 (s, 3H), 2.89
(t, J=5.66 Hz, 2H), 2.27 (s, 3H), 2.08 (s, 3H). ESI-MS calculated
for C.sub.29H.sub.27N.sub.6O.sub.4 [M+H].sup.+=523.21; Observed:
523.33.
##STR00355##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)quinolin-2-ol (Cpd. No. 142A)
CD218 (470 mg, 1.0 mmol) was dissolved in THF (18 mL). HCl aq.
solution (6 N, 30 mL) was added and the solution was heated at
75.degree. C. for 16 h. HPLC purification yielded the title
compound 370 mg (82% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 7.72
(t, J=7.73 Hz, 1H), 7.61 (d, J=7.94 Hz, 1H), 7.55 (s, 1H), 7.46 (d,
J=7.73 Hz, 1H), 7.24 (t, J=7.59 Hz, 1H), 7.14 (s, 1H), 6.64 (s,
1H), 3.38 (s, 3H), 2.99 (s, 3H), 2.68 (s, 3H), 2.08 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.22N.sub.5O.sub.3 [M+H].sup.+=452.17;
Observed: 452.92.
##STR00356##
4-(4-(2-Chloroquinolin-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7--
yl)-3,5-dimethylisoxazole (Cpd. No. 143)
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)quinolin-2-ol (Cpd. No. 142, 370 mg) and POCl.sub.3 (10 mL)
was heated at 90.degree. C. for 6 h. The volatile components were
removed on a rotary evaporator and the residue was neutralized by
NaHCO.sub.3 saturated aq. solution. The aqueous layer was extracted
with ethyl acetate and the combined organic layers were washed with
brine, dry over anhydrous sodium sulfate, and concentrated on a
rotary evaporator. The residue containing the title compound was
used for the synthesize Cpd. No. 143 without further purification.
ESI-MS calculated for C.sub.26H.sub.21.sup.35ClN.sub.5O.sub.2
[M+H].sup.+=470.14; Observed: 470.94.
##STR00357##
(2S)-4-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4-
,5-b]indol-4-yl)quinolin-2-yl)amino)butane-1,2-diol
4-(4-(2-Chloroquinolin-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-
-yl)-3,5-dimethylisoxazole (Cpd. No. 143, 100 mg, 0.2 mmol),
(2S)-4-amino-1-(triphenylmethoxy)-2-butanol (200 mg, 0.58 mmol),
K.sub.2CO.sub.3 (100 mg, 0.72 mmol), and DMSO (6 mL) were heated at
90.degree. C. for 16 h. The reaction was quenched with water. The
aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine, dry over anhydrous sodium
sulfate, and concentrated on a rotary evaporator. The residue was
dissolved in CH.sub.2Cl.sub.2 and CF.sub.3CO.sub.2H was added. The
mixture was stirred for 1 h followed by purification on preparative
HPLC to yield the title compound in 10 mg (9% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.06 (d, J=7.44 Hz, 1H), 7.89 (t, J=7.56 Hz,
1H), 7.64 (d, J=7.98 Hz, 1H), 7.47 (s, 1H), 7.50-7.40 (m, 2H), 6.62
(s, 1H), 3.90-3.70 (m, 3H), 3.58 (d, J=4.11 Hz, 2H), 3.38 (s, 3H),
2.92 (s, 3H), 2.27 (s, 3H), 2.09 (s, 3H), 2.14-2.00 (m, 1H),
2.00-1.80 (m, 1H). ESI-MS calculated for
C.sub.30H.sub.31N.sub.6O.sub.4 [M+H].sup.+=539.24; Observed:
539.83.
##STR00358##
4-(6-Methoxy-2-methyl-4-(3-(pyrrolidin-1-yl)phenyl)-9H-pyrimido[4,5-b]indo-
l-7-yl)-3,5-dimethylisoxazole
Method 42:
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isox-azole (S13, 40 mg, 0.1 mmol, 1.0 equiv.) and
3-(pyrrolidino)phenylboronic acid (70 mg, 0.3 mmol, 3.0 equiv.)
were dissolved in 1,2-dimethoxyethane (4 mL). Sodium carbonate (2.0
M in water, 2 mL) was added. The system was degassed to remove
oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (20 mg, 0.024 mmol, 0.24 equiv.)
were added and the system was degassed again and refilled with
nitrogen. The reaction mixture was heated at reflux for 16 h. The
reaction was quenched with water and extracted with ethyl acetate.
The organic layers were combined and concentrated on a rotary
evaporator. The residue was purified by reverse HPLC to afford the
title compound as a salt of CF.sub.3CO.sub.2H (30 mg, 52% yield).
.sup.1H NMR (MeOD-d4, 300 MHz): 7.59 (t, J=7.94 Hz, 1H), 7.53 (s,
1H), 7.48 (s, 1H), 7.18 (d, J=7.75 Hz, 1H), 7.10 (s, 1H), 7.00 (dd,
J=8.30, 1.98 Hz, 1H), 3.67 (s, 3H), 3.50-3.35 (m, 4H), 2.95 (s,
3H), 2.30 (s, 3H), 2.12 (s, 3H), 2.12-2.20 (m, 4H). ESI-MS
calculated for C.sub.27H.sub.28N.sub.5O.sub.2 [M+H].sup.+=454.22;
Observed: 454.68.
##STR00359##
2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N,N-dimethylaniline
Suzuki coupling of S13 and 3-(N,N-dimethylamino)phenylboronic acid,
pinacol ester using condition Method 42 afforded the title compound
as a salt of CF.sub.3CO.sub.2H (34 mg, 65% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 7.78-7.66 (m, 2H), 7.54 (s, 1H), 7.46 (d,
J=8.16 Hz, 1H), 7.29 (td, J=7.80, 0.91 Hz, 1H), 7.07 (s, 1H), 3.65
(s, 3H), 2.96 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H). ESI-MS
calculated for C.sub.25H.sub.26N.sub.5O.sub.2 [M+H].sup.+=428.21;
Observed: 428.58,
##STR00360##
4-(4-(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6-methoxy-2-methyl-9H-pyrimi-
do[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
Suzuki coupling of S13 and
5-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b-
]pyridine using condition Method 42 afforded the title compound as
a salt of CF.sub.3CO.sub.2H (10 mg, 10% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.47 (s, 1H), 7.98 (s, 1H), 7.55 (s, 1H), 7.03
(s, 1H), 3.50 (s, 3H), 2.95 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H).
ESI-MS calculated for C.sub.24H.sub.20.sup.35ClN.sub.6O.sub.2
[M+H].sup.+=459.13; Observed: 459.67,
##STR00361##
3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)aniline
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 347 mg, 1 mmol) and (3-Boc-aminophenyl) bronoic
acid (711 mg, 3 mmol), 1,2-dimethoxyethane (20 mL), and
Na.sub.2CO.sub.3 (2 M, 5 mL) were added. The system was degassed to
remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (81 mg, 0.1 mmol) was added and
the system was degassed and refilled with nitrogen. The reaction
mixture was heated at reflux for 16 h. The reaction was quenched
with water and the aqueous layer was extracted with ethyl acetate.
The organic layers were combined and the volatile components were
removed on a rotary evaporator. The residue was dissolved in
CH.sub.2Cl.sub.2 (4 mL) and CF.sub.3CO.sub.2H (4 mL) was added. The
reaction was stirred for 1 h before the volatile components were
removed on a rotary evaporator. The remaining residue was purified
by reverse HPLC to afford the title product as a salt of
CF.sub.3CO.sub.2H (80 mg, 16% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 7.74 (t, J=7.82 Hz, 1H), 7.70-7.60 (m, 2H), 7.55 (s, 1H),
7.47 (dd, J=8.04, 1.12 Hz, 1H), 7.36 (s, 1H), 3.72 (s, 3H), 2.96
(s, 3H), 2.30 (s, 3H), 2.12 (s, 3H). ESI-MS calculated for
C.sub.23H.sub.22N.sub.5O.sub.2 [M+H].sup.+=400.18; Observed:
401.00.
##STR00362##
N-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)pivalamide
3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)aniline (Cpd. No. 148, 40 mg) and pyridine (0.1 mL) were
dissolved in anhydrous THF (5 mL). To this solution,
trimethylacetic anhydride (60 mg, 0.3 mmol) was added via a syringe
and the reaction mixture was stirred at ambient temperature for 16
h. The volatile components were removed on a rotary evaporator and
the residue was purified by reverse HPLC to afford the title
product as a salt of CF.sub.3CO.sub.2H (38.2 mg, 64% yield).
.sup.1H NMR (MeOD-d4, 300 MHz): 9.52 (s, 1H), 8.61 (s, 1H),
7.84-7.68 (m, 3H), 7.54 (s, 1H), 7.51 (s, 1H), 3.70 (s, 3H), 3.52
(s, 3H), 2.96 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H), 1.32 (s, 9H).
ESI-MS calculated for C.sub.28H.sub.30N.sub.5O.sub.3
[M+H].sup.+=484.23; Observed: 484.80.
##STR00363##
4-(Methoxycarbonyl)naphthalene-1-boronic acid, pinacol ester
1-Boc-amino-4-bromonaphthalene was synthesized following preceding
procedures reported in PCT Int. Appl., 2003005999.
1-Boc-amino-4-bromonaphthalene (6.13 g, 19 mmol, 1.0 equiv.),
bis(pinacolato)diboron (9.65 g, 38 mmol, 2.0 equiv.), and potassium
acetate (5.6 g, 57 mmol, 3.0 equiv) were added to a round-bottom
flask. Anhydrous 1,4-dixoane (60 mL) was added to the flask, which
was degassed and refilled with nitrogen. Pd(dppf)Cl.sub.2 (1.0 g,
1.9 mmol, 0.1 equiv.) was added and the flask was degassed again
followed by heating at 100.degree. C. for 16 h. The reaction
mixture was cooled to room temperature and diluted by
CH.sub.2Cl.sub.2. The solution was filtered through a pad of celite
and the volatile components were removed on a rotary evaporator.
The residue was purified by flash column chromatography. The title
compound was isolated in 5.7 g (15.4 mmol, 81% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.81 (d, J=8.46 Hz, 1H), 8.06 (d, J=7.71 Hz,
1H), 7.97 (d, J=7.71 Hz, 1H), 7.83 (d, J=8.08 Hz, 1H), 7.56-7.40
(m, 2H), 7.13 (s, 1H), 1.53 (s, 9H), 1.38 (s, 12H). ESI-MS
calculated for C.sub.21H.sub.28BNNaO.sub.4 [M+Na].sup.+=392.20,
Observed: 392.42.
##STR00364##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)naphthalen-1-amine (Cpd. No. 150)
Method 40: To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.4 g, 16 mmol, 1.0 equiv.) and
4-(methoxycarbonyl)naphthalene-1-boronic acid, pinacol ester (13.75
g, 37 mmol, 2.0 equiv.), 1,2-dimethoxyethane (150 mL), and
Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was degassed
to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.3 g, 1.6 mmol, 0.1 equiv.)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
dissolved in CH.sub.2Cl.sub.2 (10 mL) and CF.sub.3CO.sub.2H (15 mL)
were added. The solution was stirred at ambient temperature for 1
h. The volatile components were removed on a rotary evaporator and
the residue was neutralized by NaHCO.sub.3 saturated solution. The
aqueous layer was extracted with ethyl acetate. The organic layers
were combined and the volatile components were removed on a rotary
evaporator. The residue was purified by flash column chromatography
to yield the title compound in 2.23 g (31% yield over two steps).
.sup.1H NMR (MeOD-d4, 300 MHz): 8.30 (d, J=8.28 Hz, 1H), 7.80 (d,
J=8.05 Hz, 1H), 7.65 (t, J=8.82 Hz, 1H), 7.58 (d, J=7.50 Hz, 1H),
7.54-7.46 (m, 1H), 7.50 (s, 1H), 7.05 (d, J=8.06 Hz, 1H), 6.33 (s,
1H), 3.20 (s, 3H), 2.97 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.27H.sub.24N.sub.5O.sub.2 [M+H].sup.+=450.19;
Observed: 450.48.
##STR00365##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(pyrrolidin-1-yl)acetamide
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 10 mg, 0.023 mmol) and
NaHCO.sub.3 (26 mg, 0.23 mmol) were dissolved in THF (4 mL). To
this solution, chloroacetyl chloride (26 mg, 0.23 mmol) was added
and the solution was stirred at ambient temperature for 16 h. To
this solution, pyrrolidine (1 mL) was added and the reaction
mixture was stirred for 12 h. The volatile components were removed
on a rotary evaporator and the residue was purified by reverse HPLC
affording the title compound as a salt of CF.sub.3CO.sub.2H (10 mg,
70% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.41 (d, J=7.89 Hz,
1H), 8.24 (d, J=7.90 Hz, 1H), 8.03 (d, J=7.87 Hz, 1H), 7.79 (t,
J=7.78 Hz, 2H), 7.67-7.59 (m, 1H), 7.52 (s, 1H), 6.17 (s, 1H), 4.54
(s, 2H), 4.00-3.80 (m, 2H), 3.40-3.20 (m, 2H), 3.16 (s, 3H), 3.01
(s, 3H), 2.26 (s, 3H), 2.40-2.10 (m, 4H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.33H.sub.33N.sub.6O.sub.3 [M+H].sup.+=561.26;
Observed: 561.67.
##STR00366##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(piperazin-1-yl)acetamide
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 45 mg, 0.1 mmol) and
NaHCO.sub.3 (160 mg, 2 mmol) were dissolved in anhydrous DMF (3
mL). To this solution, chloroacetyl chloride (113 mg, 1.0 mmol, 10.
equiv.) was added and the solution was stirred for 16 h. The
reaction mixture was diluted with water and the aqueous layer was
extracted with ethyl acetate. The organic layers were combined and
dried over anhydrous sodium sulfate. The volatile components were
removed on a rotary evaporator. The remaining residue was dissolved
in anhydrous DMF and piperazine (270 mg, 3 mmol) was added in one
portion. The reaction was stirred at ambient temperature for 16 h
before quenching with water. The aqueous layer was extracted with
ethyl acetate and the organic layers were combined, the volatile
components were removed on a rotary evaporator. The remaining
residue was purified by reverse HPLC affording the title compound
as a salt of CF.sub.3CO.sub.2H (50 mg, 74% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.33 (d, J=8.52 Hz, 1H), 8.20 (d, J=7.83 Hz,
1H), 8.04 (d, J=7.85 Hz, 1H), 7.83-7.75 (m, 2H), 7.67-7.58 (m, 1H),
7.54 (s, 1H), 6.21 (s, 1H), 3.86 (s, 2H), 3.57-3.43 (m, 4H),
3.33-3.22 (m, 4H), 3.17 (s, 3H), 3.01 (s, 3H), 2.26 (s, 3H), 2.06
(s, 3H). ESI-MS calculated for C.sub.33H.sub.34N.sub.7O.sub.3
[M+H].sup.+=576.27; Observed: 576.42.
##STR00367##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(3-oxopiperazin-1-yl)acetamide
Method 149:
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 80 mg, 0.2 mmol, 1.0
equiv.) and NaHCO.sub.3 (200 mg, 2.3 mmol, 11 equiv.) were
dissolved in anhydrous DMF (3 mL). To this solution, chloroacetyl
chloride (113 mg, 1.0 mmol, 5.0 equiv.) was added and the solution
was stirred for 16 h. The reaction mixture was diluted with water
and the aqueous layer was extracted with ethyl acetate. The organic
layers were combined and dried over anhydrous sodium sulfate. The
volatile components were removed on a rotary evaporator. The
remaining residue was dissolved in anhydrous DMF and
2-oxopiperazine (40 mg, 0.4 mmol, 2.0 equiv.) and EtN(i-Pr).sub.2
(0.2 mL) were added. The reaction was stirred at ambient
temperature for 16 h before quenching with water. The aqueous layer
was extracted with ethyl acetate and the organic layers were
combined, the volatile components were removed on a rotary
evaporator. The remaining residue was purified by reverse HPLC
affording the title compound as a salt of CF.sub.3CO.sub.2H (46 mg,
34% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.41 (d, J=8.15 Hz,
1H), 8.26 (d, J=7.87 Hz, 1H), 8.05 (d, J=7.86 Hz, 1H), 7.79 (t,
J=7.75 Hz, 2H), 7.67-7.60 (m, 1H), 7.54 (s, 1H), 6.18 (s, 1H), 4.46
(s, 2H), 4.06 (s, 2H), 3.68 (s, 4H), 3.16 (s, 3H), 3.02 (s, 3H),
2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.33H.sub.32N.sub.7O.sub.4 [M+H].sup.+=590.25; Observed:
590.75.
##STR00368##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(piperidin-4-ylamino)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and 4-amino-1-Boc piperidine afforded Boc protected title compound.
Upon treatment of CF.sub.3CO.sub.2H followed by reverse phase HPLC
purification, the title compound was isolated in 39 mg (28% over
two steps). .sup.1H NMR (MeOD-d4, 300 MHz): 8.45 (d, J=8.32 Hz,
1H), 8.25 (d, J=7.87 Hz, 1H), 8.04 (d, J=7.88 Hz, 1H), 7.78 (t,
J=7.96 Hz, 2H), 7.68-7.59 (m, 1H), 7.54 (s, 1H), 6.17 (s, 1H), 4.42
(s, 2H), 3.78-3.56 (m, 3H), 3.24-3.10 (m, 2H), 3.15 (s, 3H), 3.02
(s, 3H), 2.47 (d, J=12.26 Hz, 2H), 2.25 (s, 3H), 2.15-1.95 (m, 2H),
2.06 (s, 3H). ESI-MS calculated for C.sub.34H.sub.36N.sub.7O.sub.3
[M+H].sup.+=590.29; Observed: 590.58.
##STR00369##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(4-hydroxypiperidin-1-yl)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and 4-hydroxypiperidine afforded the title compound in 44 mg (32%
yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.42 (d, J=8.25 Hz, 1H),
8.26 (d, J=7.87 Hz, 1H), 8.05 (d, J=7.87 Hz, 1H), 7.79 (t, J=7.85
Hz, 2H), 7.67-7.60 (m, 1H), 7.54 (s, 1H), 6.18 (s, 1H), 4.45 (s,
2H), 4.20-1.00 (m, 0.5H), 3.90-3.70 (m, 1H), 3.70-3.50 (m, 2.5H),
3.16 (s, 3H), 3.02 (s, 3H), 2.30-2.10 (m, 2H), 2.25 (s, 3H),
2.10-1.80 (m, 2H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.4 [M+H].sup.+=591.27; Observed:
591.83.
##STR00370##
2-(((S)-3,4-Dihydroxybutyl)amino)-N-(4-(7-(3,5-dimethylisoxazol-4-yl)-6-me-
thoxy-2-methyl-9H-pyrimido[4,5-b]indol-4-yl)naphthalen-1-yl)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and (2S)-4-Amino-1-(triphenylmethoxy)-2-butanol afforded O-Trt
protected title compound. Upon treatment of CF.sub.3CO.sub.2H
followed by reverse phase HPLC purification, the title compound was
isolated in 16 mg (23% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.42
(d, J=8.30 Hz, 1H), 8.24 (d, J=7.84 Hz, 1H), 8.04 (d, J=7.84 Hz,
1H), 7.79 (t, J=7.87 Hz, 2H), 7.63 (t, J=7.33 Hz, 1H), 7.53 (s,
1H), 3.17 (s, 1H), 4.31 (s, 2H), 3.16 (s, 3H), 3.01 (s, 3H), 2.26
(s, 3H), 2.06 (s, 3H), 2.10-1.95 (m, 1H), 1.95-1.80 (m, 1H). ESI-MS
calculated for C.sub.33H.sub.35N.sub.6O.sub.5 [M+H].sup.+=595.27;
Observed: 595.92.
##STR00371##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-((S)-3-methylpiperazin-1-yl)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and S-1-Boc-2-methylpiperazine afforded N-Boc protected title
compound. Upon treatment of CF.sub.3CO.sub.2H followed by reverse
phase HPLC purification, the title compound was isolated in 187 mg
(90% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.39 (d, J=8.19 Hz,
1H), 8.25 (d, J=7.85 Hz, 1H), 8.07 (d, J=7.85 Hz, 1H), 7.81 (t,
J=7.77 Hz, 2H), 7.70-7.60 (m, 1H), 7.57 (s, 1H), 6.22 (s, 1H), 4.12
(s, 2H), 3.84-3.76 (m, 1H), 3.76-3.63 (m, 3H), 3.62-3.50 (m, 1H),
3.32-3.20 (m, 1H), 3.19 (s, 3H), 3.14-3.04 (m, 1H), 3.04 (s, 3H),
2.27 (s, 3H), 2.07 (s, 3H), 1.46 (d, J=6.56 Hz, 3H). ESI-MS
calculated for C.sub.34H.sub.36N.sub.7O.sub.3 [M+H].sup.+=590.29;
Observed: 590.67.
##STR00372##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-((3R,5S)-3,5-dimethylpiperazin-1-yl)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and 2,6-cis-dimethylpiperazine afforded the title compound in 60 mg
(85% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.32 (d, J=8.21 Hz,
1H), 8.21 (d, J=7.86 Hz, 1H), 8.04 (d, J=7.86 Hz, 1H), 7.82-7.75
(m, 2H), 7.63 (ddd, J=8.20, 6.98, 1.08 Hz, 1H), 7.54 (s, 1H), 6.22
(s, 1H), 3.79 (s, 2H), 3.76-3.60 (m, 2H), 3.44 (d, J=13.00 Hz, 2H),
3.17 (s, 3H), 3.01 (s, 3H), 2.68 (ddd, J=12.95, 11.18, 1.62 Hz,
2H), 2.26 (s, 3H), 2.06 (s, 3H), 1.39 (d, J=6.52 Hz, 6H). ESI-MS
calculated for C.sub.35H.sub.38N.sub.7O.sub.3 [M+H].sup.+=604.30;
Observed: 604.58.
##STR00373##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(piperidin-1-yl)acetamide
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150), chloroacetyl chloride,
and piperidine afforded the title compound in 47 mg (78% yield).
.sup.1H NMR (MeOD-d4, 300 MHz): 8.42 (d, J=8.24 Hz, 1H), 8.25 (d,
J=7.89 Hz, 1H), 8.05 (d, J=7.87 Hz, 1H), 7.79 (t, J=7.90 Hz, 2H),
7.67-7.60 (m, 1H), 7.54 (s, 1H), 6.18 (s, 1H), 4.42 (s, 2H),
3.80-3.68 (m, 2H), 3.30-3.14 (m, 2H), 3.16 (s, 3H), 3.02 (s, 3H),
2.25 (s, 3H), 2.06 (s, 3H), 2.08-1.80 (m, 5H), 1.70-1.50 (m, 1H).
ESI-MS calculated for C.sub.34H.sub.35N.sub.6O.sub.3
[M+H].sup.+=575.28; Observed: 575.48.
##STR00374##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N,N-bis(2-morpholinoethyl)naphthalen-1-amine
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 27 mg, 0.05 mmol) and
3-Morpholinopropanal-HCl (18 mg, 0.1 mmol) were dissolved in THF (5
mL). AcOH (0.1 mL) and NaBH(OAc).sub.3 (50 mg, 0.2 mmol) were added
and the mixture was stirred for 16 h. The reaction was quenched
with water and extracted with ethyl acetate. The organic layers
were combined and removed on a rotary evaporator. The residue was
purified by reverse HPLC affording the title compound as a salt of
CF.sub.3CO.sub.2H (14 mg, 36% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.54 (d, J=8.28 Hz, 1H), 8.02 (d, J=7.79 Hz, 1H), 7.84-7.74
(m, 3H), 7.66-7.58 (m, 1H), 7.54 (s, 1H), 6.25 (s, 1H), 410-3.80
(m, 12H), 3.60-3.40 (m, 6H), 3.19 (s, 3H), 3.02 (s, 3H), 2.27 (s,
3H), 2.07 (s, 3H). ESI-MS calculated for
C.sub.39H.sub.46N.sub.7O.sub.4 [M+H].sup.+=676.36; Observed:
676.75.
##STR00375##
4-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)naphthalen-1-yl)amino)-4-oxobutanoic acid (Cpd. No.
161)
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 120 mg, 0.3 mmol),
succinic anhydride (60 mg, 0.6 mmol), and pyridine (2 mL) were
dissolved in anhydrous DMF (5 mL) and the mixture was heated at
70.degree. C. for 16 h. The reaction mixture was concentrated on a
rotary evaporator and purified by reverse phase HPLC affording the
title compound in 120 mg (60% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.42 (d, J=8.75 Hz, 1H), 8.10 (d, J=7.64 Hz, 1H), 8.00 (d,
J=7.77 Hz, 1H), 7.80-7.70 (m, 2H), 7.65-7.56 (m, 1H), 1.53 (s, 1H),
6.20 (s, 1H), 3.17 (s, 3H), 3.01 (s, 3H), 2.93 (t, J=6.23 Hz, 2H),
2.79 (t, J=6.23 Hz, 2H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.31H.sub.28N.sub.5O.sub.5 [M+H].sup.+=550.21;
Observed: 550.50.
##STR00376##
N1-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)naphthalen-1-yl)-N4-ethylsuccinamide
4-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b-
]indol-4-yl)naphthalen-1-yl)amino)-4-oxobutanoic acid (Cpd. No.
161, 60 mg, 0.1 mmol), EDCI-HCl (100 mg, 0.5 mmol), HOBt-H.sub.2O
(70 mg, 0.5 mmol), and anhydrous DMF (2.5 mL) were added to a
round-bottom flask. EtNH.sub.2 (2 M in THF, 1 mL) was added
followed by addition of via a syringe and the reaction mixture was
stirred for 16 h at ambient temperature. The reaction mixture was
purified by reverse HPLC affording the title compound as a salt of
CF.sub.3CO.sub.2H (22 mg, 33% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.42 (d, J=8.32 Hz, 1H), 8.14 (d, J=7.86 Hz, 1H), 7.99 (d,
J=7.86 Hz, 1H), 7.76 (t, J=7.57 Hz, 2H), 7.64-7.56 (m, 1H), 7.52
(s, 1H), 6.21 (s, 1H), 3.25 (q, J=7.33 Hz, 2H), 3.17 (s, 3H), 3.01
(s, 3H), 2.93 (t, J=6.80 Hz, 2H), 2.68 (t, J=6.80 Hz, 2H), 2.26 (s,
3H), 2.06 (s, 3H), 1.14 (t, J=7.33 Hz, 3H). ESI-MS calculated for
C.sub.33H.sub.33N.sub.6O.sub.4 [M+H].sup.+=577.26; Observed:
577.92.
##STR00377##
tert-Butyl
(2-(tert-butyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)carbamate (CE128)
4-Bromo-2-(1,1-dimethylethyl)aniline (0.95 g, 4.2 mmol) and Boc
anhydride (1.20 g, 5.46 mmol) were dissolved in anhydrous toluene
(10 mL) and the solution was heated at 90.degree. C. for 24 h. The
mixture was purified by flash column chromatography to yield
tert-butyl (4-bromo-2-(tert-butyl)phenyl)carbamate (2.42 g,
contaminated with Boc.sub.2O). tert-Butyl
(4-bromo-2-(tert-butyl)phenyl)carbamate (2.42 g from previous step,
view as 4.16 mmol), bis(pinacolato)diboron (2.13 g, 8.4 mmol, 2.0
equiv.), and potassium acetate (1.6 g, 16 mmol, 4.0 equiv.) were
added to a round-bottom flask Anhydrous 1,4-dixoane (20 mL) was
added via a syringe and the flask was degassed and refilled with
nitrogen. Pd(dppf)Cl.sub.2 (322 mg, 0.46 mmol, 0.1 equiv.) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to ambient
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound was isolated in 2.0 g
(contaminated with boronic acid pinacol ester). This material was
used for preparation of Cpd. No. 162 without further purification.
.sup.1H NMR (MeOD-d4, 300 MHz): 7.77 (s, 1H), 7.70 (d, J=7.98 Hz,
1H), 7.63 (d, J=7.98 Hz, 1H), 6.54 (s, 1H), 1.49 (s, 9H), 1.42 (s,
9H), 1.32 (s, 12H). ESI-MS calculated for
C.sub.21H.sub.34BNNaO.sub.4 [M+Na].sup.+=398.25; Observed:
398.50.
##STR00378##
2-(tert-Butyl)-4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyri-
mido[4,5-b]indol-4-yl)aniline (Cpd. No. 162)
Using same protocol similar to Method 40: Suzuki coupling of S13
(800 mg, 2.16 mmol) and tert-Butyl
(2-(tert-butyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)car-
bamate (CE128, 2.0 g) followed by CF.sub.3CO.sub.2H-promoted
deprotection of Boc group yielded the title compound after flash
column chromatography (510 mg, 52% yield). .sup.1H NMR (MeOD-d4,
300 MHz): 7.75 (d, J=1.87 Hz, 1H), 7.51 (dd, J=8.21, 1.85 Hz, 1H),
7.45 (s, 1H), 7.26 (s, 1H), 6.87 (d, J=8.20 Hz, 1H), 3.63 (s, 3H),
2.69 (s, 3H), 2.25 (s, 3H), 2.01 (s, 3H), 1.41 (s, 9H). ESI-MS
calculated for C.sub.27H.sub.30N.sub.5O.sub.2 [M+H].sup.+=456.24;
Observed: 456.67.
##STR00379##
N-(2-(tert-Butyl)-4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-p-
yrimido[4,5-b]indol-4-yl)phenyl)-2-((3R,5S)-3,5-dimethylpiperazin-1-yl)ace-
tamide
42-(tert-Butyl)-4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-py-
rimido[4,5-b]indol-4-yl)aniline(Cpd. No. 162, 70 mg, 0.2 mmol, 1.0
equiv.) and NaHCO.sub.3 (170 mg, 2.0 mmol, 10 equiv.) were
dissolved in anhydrous THF (6 mL). To this solution, chloroacetyl
chloride (120 mg, 1.0 mmol, 5.0 equiv.) was added and the solution
was stirred for 16 h. The reaction mixture was diluted with water
and the aqueous layer was extracted with ethyl acetate. The organic
layers were combined and dried over anhydrous sodium sulfate. The
volatile components were removed on a rotary evaporator. The
remaining residue was dissolved in anhydrous DMF and
2,6-cis-dimethylpiperazine (66 mg, 0.6 mmol, 3.0 equiv.) and
EtN(iPr.sub.2) (0.2 mL) were added. The reaction was stirred at
ambient temperature for 16 h before quenching with water. The
aqueous layer was extracted with ethyl acetate and the organic
layers were combined, the volatile components were removed on a
rotary evaporator. The remaining residue was purified by reverse
phase HPLC affording the title compound as a salt of
CF.sub.3CO.sub.2H (86 mg, 60% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.10 (d, J=1.74 Hz, 1H), 8.05 (d, J=8.24 Hz, 1H), 7.93 (dd,
J=8.24, 1.81 Hz, 1H), 7.57 (s, 1H), 7.35 (s, 1H), 3.97 (s, 2H),
3.71 (s, 3H), 3.64-3.50 (m, 2H), 3.40-3.32 (m, 2H), 2.97 (s, 3H),
2.64 (t, J=12.16 Hz, 2H), 2.31 (s, 3H), 2.13 (s, 3H), 1.55 (s, 9H),
1.38 (d, J=6.55 Hz, 6H). ESI-MS calculated for
C.sub.35H.sub.44N.sub.7O.sub.3 [M+H].sup.+=610.35; Observed:
610.58.
##STR00380##
Methyl
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-
-b]indol-4-yl)-1-naphthoate (Cpd. No. 164)
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 1.71 g, 5.0 mmol, 1.0 equiv.) and methyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoate (3.0
g, 10 mmol, 2.0 equiv.) were dissolved in 1,2-dimethoxyethane (60
mL). Sodium carbonate (2.0 M in water, 20 mL) was added. The system
was degassed to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (408 mg, 0.5 mmol, 0.1 equiv.)
was added and the flask was degassed again and refilled with
nitrogen. The reaction mixture was heated at reflux for 16 h. The
reaction was quenched with water and extracted with ethyl acetate.
The organic layers were combined and removed on a rotary
evaporator. The residue was purified by flash column chromatography
to yield the title compound (1.04 g, 42% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz): 11.73n (s, 1H), 9.09 (d, J=8.74 Hz, 1H),
8.39 (d, J=7.49 Hz, 1H), 7.84 (d, J=7.57 Hz, 2H), 7.72-7.65 (m,
1H), 7.53-7.46 (m 1H), 7.31 (s, 1H), 6.22 (s, 1H), 4.08 (s, 3H),
3.20 (s, 3H), 3.07 (s, 3H), 2.29 (s, 3H), 2.13 (s, 3H). ESI-MS
calculated for C.sub.29H.sub.25N.sub.4O.sub.4 [M+H].sup.+=493.19;
Observed: 493.50.
##STR00381##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-1-naphthoic acid (Cpd. No. 165)
Methyl
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,-
5-b]indol-4-yl)-1-naphthoate (Cpd. No. 164, 107 mg, 0.22 mmol) was
dissolved in THF (5 mL) and water (5 mL). LiOH--H.sub.2O (90 mg,
2.0 mmol, 10.0 equiv.) was added and solution was stirred for 16 h.
The reaction mixture was extracted with ethyl acetate.
Subsequently, the aqueous layer was neutralized to pH=2 and was
extracted with ethyl acetate. The organic extracts of acidic
aqueous solution were combined and concentrated on a rotary
evaporator. The remaining residue was freeze-dried to yield the
title compound in 100 mg (>90% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 9.13 (d, J=8.60 Hz, 1H), 8.47 (d, J=7.47 Hz, 1H), 8.07 (d,
J=7.48 Hz, 1H), 7.84-7.74 (m, 2H), 7.61 (t, J=7.63), 7.55 (s, 1H),
6.13 (s, 1H), 3.16 (s, 3H), 3.03 (s, 3H), 2.24 (s, 3H), 2.04 (s,
3H). ESI-MS calculated for C.sub.28H.sub.23N.sub.4O.sub.4
[M+H].sup.+=479.17; Observed: 479.42.
##STR00382##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-morpholinoethyl)-1-naphthamide
Method 64 (amide condensation):
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 20 mg, 0.05 mmol),
EDCI-HCl (100 mg, 0.5 mmol), and HOBt-H.sub.2O (70 mg, 0.5 mmol)
were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.1 mL) was
added followed by addition of DMF (2.5 mL). 2-Morpholinylethylamine
(70 mg, 0.5 mmol) was added and the reaction mixture was stirred
for 12 h. The reaction was quenched with NaHCO.sub.3 saturated
solution and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were concentrated on a rotary
evaporator. The remaining residue was purified by reverse phase
HPLC affording the title compound as a salt of CF.sub.3CO.sub.2H
(20 mg, 69% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.55 (d, J=8.52
Hz, 1H), 8.10-8.02 (m, 2H), 7.84-7.74 (m, 2H), 7.68-7.60 (m, 1H),
7.54 (s, 1H), 6.15 (s, 1H), 4.20-4.00 (m, 2H), 4.00-3.60 (m, 8H),
3.97 (t, J=5.72 Hz, 2H), 3.57 (t, J=6.24 Hz, 2H), 3.17 (s, 3H),
3.02 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.4 [M+H].sup.+=591.27; Observed:
591.58.
##STR00383##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-ureidoethyl)-1-naphthamide
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 20 mg) and
(2-Amino-ethyl)-urea-HCl (20 mg) afforded the title compound in 16
mg (57% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.49 (d, J=8.45 Hz,
1H), 8.03 (d, J=7.33 Hz, 1H), 7.97 (d, J=7.33 Hz, 1H), 7.82-7.73
(m, 2H), 7.65-7.58 (m, 1H), 7.53 (s, 1H), 6.17 (s, 1H), 3.63 (t,
J=5.63 Hz, 2H), 3.47 (t, J=5.97 Hz, 1H), 3.19 (s, 3H), 3.01 (s,
3H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.31H.sub.30N.sub.7O.sub.4 [M+H].sup.+=564.24; Observed:
564.50.
##STR00384##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(piperazin-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 20 mg) and piperazine (63
mg) afforded the title compound in 8 mg (30% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.20-8.04 (m, 2H), 8.00-7.78 (m, 2H), 7.72-7.60
(m, 1H), 7.58-7.50 (m, 1H), 6.26-6.14 (m, 1H), 4.40-4.10 (m, 2H),
3.80-3.40 (m, 4H), 3.40-3.20 (m, 2H), 3.19 (s, 3H), 3.03 (s, 3H),
2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.31N.sub.6O.sub.3 [M+H].sup.+=547.25; Observed:
547.67.
##STR00385##
Methyl
3-(4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[-
4,5-b]indol-4-yl)-1-naphthamido)propanoate
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 30 mg) and .beta.-alanine
methyl ester (28 mg) afforded the title compound. The crude was
used in the next step without further purification.
##STR00386##
3-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)-1-naphthamido)propanoic acid
Methyl
3-(4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido-
[4,5-b]indol-4-yl)-1-naphthamido)propanoate (Cpd. No. 169) was
dissolved in THF--H.sub.2O (1:1). LiOH--H.sub.2O (10 equiv.) was
added and the reaction was stirred at ambient temperature for 16 h.
Volatile components were removed on a rotary evaporator and the
residues was purified by on a reverse phase HPLC affording the
title compound (22 mg, 34% yield). .sup.1H NMR (MeOD-d4, 300 MHz):
8.46 (d, J=8.49 Hz, 1H), 8.02 (d, J=7.32 Hz, 1H), 7.92 (d, J=7.32
Hz, 1H), 7.83-7.72 (m, 2H), 7.65-7.57 (m, 1H), 7.52 (s, 1H), 6.15
(s, 1H), 3.80 (t, J=6.65 Hz, 2H), 3.18 (s, 3H), 3.01 (s, 3H), 2.78
(t, J=6.65 Hz, 2H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS calculated
for C.sub.31H.sub.28N.sub.5O.sub.5 [M+H].sup.+=550.21; Observed:
550.33.
##STR00387##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-morpholinopiperidine-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 40 mg) and
4-morpholinopiperidine (34 mg) afforded the title compound in 50 mg
(78% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.22-8.02 (m, 2H),
8.00-7.78 (m, 3H), 7.72-7.62 (m, 1H), 7.60-7.56 (m, 1H), 6.26-6.16
(m, 1H), 5.20-5.06 (m, 1H), 4.40-4.00 (m, 2H), 4.00-3.55 (m, 2H),
3.54-3.45 (m, 4H), 3.40-3.00 (m, 4H), 3.22 (s, 3H), 3.05 (s, 3H),
2.60-2.40 (m, 1H), 2.28 (s, 3H), 2.20-1.80 (m, 2.5H), 2.08 (s, 3H),
1.70-1.50 (m, 0.5H). ESI-MS calculated for
C.sub.37H.sub.39N.sub.6O.sub.4 [M+H].sup.+=631.30; Observed:
631.83.
##STR00388##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(3,3,4-trimethylpiperazine-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 20 mg) and
1,2,2-trimethylpiperidine (20 mg) afforded the title compound in 12
mg (40% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.20-8.00 (m, 2H),
8.00-7.80 (m, 3H), 7.70-7.60 (m, 1H), 7.60-7.50 (m, 1H), 6.40-6.10
(m, 1H), 3.80-3.40 (m, 4H), 3.40-3.10 (m, 2H), 3.02 (s, 3H), 2.90
(s, 3H), 2.26 (s, 3H), 2.07 (s, 3H), 1.70-1.50 (m, 3H), 1.40-1.00
(m, 3H). ESI-MS calculated for C.sub.35H.sub.37N.sub.6O.sub.3
[M+H].sup.+=589.29; Observed: 589.83.
##STR00389##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)((S)-3,4-dimethylpiperazin-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 160 mg) and
(S)-1,2-dimethyl-piperazine (224 mg) afforded the title compound in
150 mg (55% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.30-8.00 (m,
2H), 8.00-7.75 (m, 3H), 7.75-7.60 (m, 1H), 7.45 (s, 1H), 6.40-6.10
(m, 1H), 3.90-3.20 (m, 6H), 3.32 (s, 6H), 3.20 (s, 3H), 2.27 (s,
3H), 2.08 (s, 3H), 1.59 (d, J=6.30 Hz, 1.5H), 1.40-1.10 (m, 1.5H).
ESI-MS calculated for C.sub.34H.sub.35N.sub.6O.sub.3
[M+H].sup.+=575.28; Observed: 575.83.
##STR00390##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)((3R,5S)-3,5-dimethylpiperazin-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 160 mg) and
cis-2,6-dimethyl-piperazine (160 mg) afforded the title compound in
138 mg (52% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.30-8.00 (m,
2H), 8.00-7.70 (m, 3H), 7.70-7.60 (m, 1H), 7.60-7.50 (m, 1H),
6.50-6.10 (m, 1H), 5.20-5.00 (m, 1H), 3.80-3.20 (m, 4H), 3.19 (s,
3H), 3.03 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H), 1.56-1.46 (m, 3H),
1.24-1.10 (m, 3H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.3 [M+H].sup.+=575.28; Observed:
575.75.
##STR00391##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 160 mg) and
1-(2-Hydroxyethyl)piperazine (140 mg) afforded the title compound
in 172 mg (62% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.30-8.05
(m, 2H), 8.00-7.75 (m, 3H), 7.70-7.60 (m, 1H), 7.57 (s, 1H), 6.21
(s, 1H), 3.96 (t, J=4.39 Hz, 2H), 3.80-3.40 (m, 8H), 3.42 (t,
J=4.39 Hz, 2H), 3.19 (s, 3H), 3.04 (s, 3H), 2.23 (s, 3H), 2.04 (s,
3H). ESI-MS calculated for C.sub.34H.sub.35N.sub.6O.sub.4
[M+H].sup.+=591.27; Observed: 591.50.
##STR00392##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)-1-naphthamide
Using amide condensation condition Method 64, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 40 mg) and
1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine dihydrochloride (50
mg) afforded the title compound in 87 mg (>90% yield). .sup.1H
NMR (MeOD-d4, 300 MHz): 8.45 (d, J=8.46 Hz, 1H), 8.05 (d, J=7.20
Hz, 1H), 7.96 (d, J=7.34 Hz, 1H), 7.85-7.73 (m, 2H), 7.62 (ddd,
J=8.23, 7.10, 1.03 Hz, 1H), 7.55 (s, 1H), 6.15 (s, 1H), 4.44-4.30
(m, 1H), 4.09 (dd, J=11.26, 3.87 Hz, 2H), 3.76 (d, J=12.35 Hz, 2H),
3.60-3.40 (m, 4H), 3.34-3.20 (m, 1H), 3.17 (s, 3H), 3.02 (s, 3H),
2.53-2.40 (m, 2H), 2.25 (s, 3H), 2.18-1.96 (m, 4H), 2.06 (s, 3H),
1.90-1.72 (m, 2H). ESI-MS calculated for
C.sub.38H.sub.41N.sub.6O.sub.4 [M+H].sup.+=645.32; Observed:
645.58.
##STR00393##
N--((S)-3,4-Dihydroxybutyl)-4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-m-
ethyl-9H-pyrimido[4,5-b]indol-4-yl)-1-naphthamide
Method 77:
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 20 mg, 0.05 mmol),
EDCI-HCl (48 mg, 0.25 mmol), and HOBt-H.sub.2O (34 mg, 0.25 mmol)
were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.1 mL) was
added followed by addition of DMF (2.5 mL) via syringes.
(2S)-4-Amino-1-(triphenylmethoxy)-2-butanol (52 mg, 0.15 mmol) was
added and the reaction mixture was stirred for 16 h. The reaction
was quenched with NaHCO.sub.3 saturated solution and the aqueous
layer was extracted with ethyl acetate. The combined organic layers
were concentrated on a rotary evaporator. The residue was dissolved
in CH.sub.2Cl.sub.2 (4 mL) and CF.sub.3CO.sub.2H (4 mL) was added
and the mixture was stirred for 1 h before purification on a
reverse phase HPLC affording the title compound as a salt of
CF.sub.3CO.sub.2H (19 mg, 68% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.46 (d, J=8.39 Hz, 1H), 8.04 (d, J=7.32 Hz, 1H), 7.95 (d,
J=7.32 Hz, 1H), 7.84-7.74 (m, 2H), 7.66-7.58 (m, 1H), 7.54 (s, 1H),
6.17 (s, 1H), 3.86-3.75 (m, 1H), 3.75-3.66 (m, 2H), 3.56 (d, J=5.55
Hz, 2H), 3.19 (s, 3H), 3.02 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H),
2.10-1.90 (m, 1H), 1.90-1.70 (m, 1H). ESI-MS calculated for
C.sub.32H.sub.32N.sub.5O.sub.5 [M+H].sup.+=566.24; Observed:
566.75.
##STR00394##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)((S)-3-methylpiperazin-1-yl)methanone
Using amide condensation condition Method 77, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 40 mg) and
(S)-1-Boc-2-methyl-piperazine (60 mg) followed by
CF.sub.3CO.sub.2H-promoted deprotection of Boc group afforded the
title compound in 27 mg (41% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.28-8.00 (m, 2H), 8.00-7.74 (m, 2H), 7.70-7.60 (m, 1H), 7.53
(s, 1H), 6.40-6.10 (m, 1H), 3.80-3.40 (m, 4H), 3.40-3.10 (m, 3H),
3.19 (s, 3H), 3.02 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H), 1.51 (d,
J=5.55 Hz, 1.5H), 1.16 (d, J=5.84 Hz, 1.5H). ESI-MS calculated for
C.sub.33H.sub.33N.sub.6O.sub.3 [M+H].sup.+=561.26; Observed:
561.58.
##STR00395##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(piperidin-4-yl)-1-naphthamide
Using amide condensation condition Method 77, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 40 mg) and
1-Boc-4-amino-piperidine (60 mg) followed by
CF.sub.3CO.sub.2H-promoted deprotection of Boc group afforded the
title compound in 34 mg (51% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 9.15 (d, J=6.96 Hz, 1H), 8.44 (d, J=8.55 Hz, 1H), 8.04 (d,
J=7.31 Hz, 1h), 7.96 (d, J=7.31 Hz, 1H), 7.81 (d, J=8.28 Hz, 1H),
7.76 (d, J=8.42 Hz, 1H), 7.62 (t, J=7.63 Hz, 1H), 7.54 (s, 1H),
6.15 (s, 1H), 4.50-4.30 (m, 1H), 3.55 (d, J=12.83 Hz, 2H),
3.30-3.14 (m, 2H), 3.18 (s, 3H), 3.02 (s, 3H), 2.44-2.30 (m, 2H),
2.26 (s, 3H), 2.06 (s, 3H), 2.00-1.86 (m, 2H). ESI-MS calculated
for C.sub.33H.sub.33N.sub.6O.sub.3 [M+H].sup.+=561.26; Observed:
561.58.
##STR00396##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(3,3-dimethylpiperazin-1-yl)methanone
Using amide condensation condition Method 77, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 160 mg) and
1-Boc-2,2-dimethylpiperidine (260 mg) followed by
CF.sub.3CO.sub.2H-promoted deprotection of Boc group afforded the
title compound in 150 mg (56% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.30-8.10 (m, 2H), 8.10-7.75 (m, 3H), 7.70-7.60 (m, 1H),
7.60-7.50 (m, 1H), 6.40-6.20 (m, 1H), 4.40-4.20 (m, 1.4H),
4.00-3.80 (m, 0.6H), 3.80-3.30 (m, 4H), 3.21 (s, 3H), 3.04 (s, 3H),
2.27 (s, 3H), 2.08 (s, 3H), 1.62 (d, J=3.58 Hz, 3H), 1.38 (s,
1.5H), 1.30-1.20 (m, 1.5H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.3 [M+H].sup.+=575.28; Observed:
575.75.
##STR00397##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)((R)-3-(hydroxymethyl)piperazin-1-yl)methanone
(Cpd. No. 181)
Using amide condensation condition Method 77, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-1-naphthoic acid (Cpd. No. 165, 100 mg) and
(R)-1-Boc-2-hydroxymethylpiperazine (80 mg) followed by
CF.sub.3CO.sub.2H-promoted deprotection of Boc group afforded the
title compound in 92 mg (59% yield). .sup.1H NMR (MeOD-d4, 300
MHz): 8.30-8.05 (m, 2H), 8.05-7.75 (m, 3H), 7.75-7.60 (m, 1H), 7.56
(s, 1H), 6.40-6.40 (m, 1H), 5.10-4.90 (m, 1H), 4.10-3.40 (m, 8H),
3.18 (s, 3H), 3.04 (s, 3H), 2.24 (s, 3H), 2.05 (s, 3H). ESI-MS
calculated for C.sub.33H.sub.33N.sub.6O.sub.4 [M+H].sup.+=577.26;
Observed: 577.83.
##STR00398##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)((R)-3-(hydroxymethyl)-4-methylpiperazin-1-yl)meth-
anone (Cpd. No. 182)
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]in-
dol-4-yl)naphthalen-1-yl)((R)-3-(hydroxymethyl)piperazin-1-yl)methanone
(Co, 20 mg), paraformaldehyde (30 mg), acetic acid (0.05 mL), and
1,2-dichloroethane (4 mL) were placed in a round-bottom flask.
Sodium triacetoxyborohydride (400 mg) was added in one portion and
the mixture was stirred at ambient temperature for 16 h. Water was
added and the aqueous layer was extracted with ethyl acetate and
CH.sub.2Cl.sub.2. The combined organic layers were concentrated on
a rotary evaporator. The residue was purified on a reverse phase
HPLC affording the title compound as a salt of CF.sub.3CO.sub.2H
(11 mg, 52% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.30-8.00 (m,
2H), 8.00-7.75 (m, 3H), 7.75-7.60 (m, 1H), 7.53 (s, 1H), 6.50-6.10
(m, 1H), 5.10-4.90 (m, 1H), 4.30-3.20 (m, 8H), 3.19 (s, 3H), 3.04
(s, 3H), 3.02 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS
calculated for C.sub.34H.sub.35N.sub.6O.sub.4 [M+H].sup.+=591.27;
Observed: 591.67.
##STR00399##
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)propan-2-ol (free amine)
S13 (40 mg, 0.1 mmol, 1.0 equiv.) and
3-(2-hydroxy-2-propanyl)phenylboronic acid pinacol ester (90 mg,
0.3 mmol, 3.0 equiv.) were dissolved in 1,2-dimethoxyethane (4 mL).
Sodium carbonate (2.0 M in water, 2 mL) was added. The system was
degassed to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (20 mg, 0.024 mmol, 0.24 equiv.)
were added and the system was degassed again and refilled with
nitrogen. The reaction mixture was heated at reflux for 16 h. The
reaction was quenched with water and extracted with ethyl acetate.
The organic layers were combined and removed on a rotary
evaporator. The residue was purified by reverse phase HPLC. The
HPLC eluents containing the title compound was neutralized with
ammonia solution and extracted with ethyl acetate to afford the
title compound as free amine (10 mg, 22% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 8.12 (s, 1H), 7.90-7.60 (m, 2H), 7.71 (t,
J=7.66 Hz, 1H), 7.50 (s, 1H), 7.31 (s, 1H), 3.66 (s, 3H), 2.89 (s,
3H), 2.30 (s, 3H), 2.12 (s, 3H), 1.63 (s, 6H). ESI-MS calculated
for C.sub.26H.sub.27N.sub.4O.sub.3 [M+H].sup.+=443.21; Observed:
443.72.
##STR00400##
2-(tert-Butyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(CE103)
4-Bromo-2-(tert-butyl)pyridine (1.0 g, 4.6 mmol, 1.0 equiv.) was
dissolved in anhydrous THF (20 mL). The solution was cooled to
-78.degree. C. for 15 min before BuLi (3.7 mL, 2.5 M in THF, 9.2
mmol, 2.0 equiv.) was added via a syringe. The reaction solution
was stirred at -78.degree. C. for 30 min and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.37 g, 7.36
mmol, 1.6 equiv.) was added via a syringe. The reaction was stirred
at -78.degree. C. for 3 h before quenching with saturated
NH.sub.4Cl aqueous solution. The aqueous layer was extracted with
ethyl acetate and the combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and concentrated on a
rotary evaporator. The remaining residue was purified by flash
column chromatography to yield the title compound in 70 mg (6%
yield). .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.51 (d, J=4.68 Hz, 1H),
7.61 (s, 1H), 7.36 (d, J=4.68 Hz, 1H), 1.31 (s, 9H), 1.26 (s, 12H).
.sup.13C NMR (CDCl.sub.3, 75 MHz): 168.53, 148.06, 125.78, 124.07,
84.34, 37.42, 30.32, 24.90. ESI-MS calculated for
C.sub.15H.sub.25BNO.sub.2 [M+H].sup.+=262.20; Observed: 262.42.
##STR00401##
4-(4-(2-(tert-Butyl)pyridin-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-7-yl)-3,5-dimethylisoxazole
Suzuki coupling of S13 (273 mg, 0.80 mmol) and
2-(tert-butyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(CE103, 440 mg, 1.68 mmol) using condition Method 42 followed by
flash column chromatography afforded the title compound in 180 mg
(51% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 12.62 (s, 1H), 8.81
(d, J=4.84 Hz, 1H), 7.85 (s, 1H), 7.60 (d, J=4.65 Hz, 1H), 7.34 (s,
1H), 7.28 (s, 1H), 3.66 (s, 3H), 2.84 (s, 2H), 2.31 (s, 3H), 2.17
(s, 3H), 1.43 (s, 9H). ESI-MS calculated for
C.sub.26H.sub.28N.sub.5O.sub.2 [M+H].sup.+=442.22; Observed:
442.50.
##STR00402##
2-(2-((tert-Butyldimethylsilyl)oxy)propan-2-yl)-4-(4,4,5,5-tetramethyl-1,3-
,2-dioxaborolan-2-yl)pyridine (CE81)
2-(4-Bromopyridin-2-yl)propan-2-ol (0.57 g, 3.0 mmol), TBS-Cl (1.35
g, 9.0 mmol), and imidazole (816 mg, 12 mmol) were dissolved in
anhydrous DMF (20 mL). The solution was heated at reflux for 3 days
before quenching with water. The aqueous layer was extracted with
ethyl acetate and the combined organic layers were washed with
brine, dry over anhydrous sodium sulfate, and concentrated on a
rotary evaporator. The residue was purified by flash column
chromatography to yield
4-bromo-2-(2-((tert-butyldimethylsilyl)oxy)propan-2-yl)pyridine
(0.46 g, 46% yield).
4-Bromo-2-(2-((tert-butyldimethylsilyl)oxy)propan-2-yl)pyridine
(0.46 g, 1.4 mmol, 1.0 equiv.), bis(pinacolato)diboron (0.711 g,
2.8 mmol, 2.0 equiv.), and potassium acetate (0.549 g, 5.6 mmol,
4.0 equiv) were added to a round-bottom flask Anhydrous 1,4-dixoane
(15 mL) was added and the flask was degassed and refilled with
nitrogen. Pd(dppf)Cl.sub.2 (98 mg, 0.14 mmol, 0.1 equiv.) was added
and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to ambient
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography to yield the title compound in 0.60 g (1.3
mmol, 90% yield). .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.52 (d,
J=4.74 Hz, 1H), 8.13 (t, J=0.98 Hz, 1H), 7.43 (dd, J=4.73, 1.08 Hz,
1H), 1.60 (s, 6H), 1.33 (s, 12H), 0.96 (s, 9H), 0.07 (s, 6H).
ESI-MS calculated for C.sub.20H.sub.37BNO.sub.3Si
[M+H].sup.+=378.26; Observed: 378.33.
##STR00403##
4-(4-(2-(2-((tert-Butyldimethylsilyl)oxy)propan-2-yl)pyridin-4-yl)-6-metho-
xy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
(CE83)
Suzuki coupling of S13 (240 mg, 0.7 mmol) and
2-(2-((tert-butyldimethylsilyl)oxy)propan-2-yl)-4-(4,4,5,5-tetramethyl-1,-
3,2-dioxaborolan-2-yl)pyridine (CE81, 0.62 g, 1.4 mmol) using
condition Method 42 followed by flash column chromatography
afforded the title compound in 0.337 mg (87% yield). .sup.1H NMR
(MeOD-d.sub.4 and CDCl.sub.3, 300 MHz): 8.77 (d, J=4.99 Hz, 1H),
8.32 (s, 1H), 7.70 (dd, J=4.98, 1.48 Hz, 1H), 7.32 (s, 1H), 7.28
(s, 1H), 3.67 (s, 3H), 2.87 (s, 3H), 2.32 (s, 3H), 2.16 (s, 3H),
1.70 (s, 6H), 0.77 (s, 9H), 0.11 (s, 6H). ESI-MS calculated for
C31H40N5O3Si [M+H].sup.+=558.29; Observed: 558.76.
##STR00404##
2-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)pyridin-2-yl)propan-2-ol
4-(4-(2-(2-((tert-Butyldimethylsilyl)oxy)propan-2-yl)pyridin-4-yl)-6-meth-
oxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
(CE83, 0.337 g) was dissolved in 20 mL concentrated HCl and the
mixture was stirred at room temperature for 24 h. The reaction
mixture was purified by reverse phase HPLC affording the title
compound as a salt of CF.sub.3CO.sub.2H (60 mg, 18% yield). .sup.1H
NMR (MeOD-d4, 300 MHz): 8.79 (d, J=5.06 Hz, 1H), 8.28 (s, 1H), 7.76
(dd, J=5.03, 1.57 Hz, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 3.71 (s,
3H), 2.79 (s, 3H), 2.30 (s, 3H), 2.13 (s, 3H), 1.65 (s, 6H). ESI-MS
calculated for C.sub.25H.sub.26N.sub.5O.sub.3 [M+H].sup.+=444.20;
Observed: 444.92.
##STR00405##
2-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)pyridin-2-yl)-2-methylpropanenitrile
Suzuki coupling of S13 (342 mg, 1.0 mmol) and
2-methyl-2-[4-(tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl]propaneni-
trile (0.5 g, 1.82 mmol) using condition Method 42 followed by
reverse phase HPLC purification afforded the title compound as a
salt of CF.sub.3CO.sub.2H (227 mg, 41% yield). .sup.1H NMR
(MeOD-d4, 300 MHz): 9.06 (d, J=4.99 Hz, 1H), 8.25 (s, 1H), 8.00 (d,
J=4.99, 1.47 Hz, 1H), 7.57 (s, 1H), 7.22 (s, 1H), 3.73 (s, 3H),
2.98 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H), 1.88 (s, 6H). ESI-MS
calculated for C.sub.26H.sub.25N.sub.6O.sub.2 [M+H].sup.+=453.20;
Observed: 453.67.
##STR00406##
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)-2-methylpropanoic acid (Cpd. No. 187)
Suzuki coupling of S13 (342 mg, 1.0 mmol) and
3-borono-a,a-dimethyl-benzeneacetic acid (0.42 g, 2.0 mmol) using
condition Method 42 followed by reverse phase HPLC purification
afforded the title compound as a salt of CF.sub.3CO.sub.2H (88 mg,
15% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 8.02 (s, 1H), 7.93-7.77
(m, 2H), 7.57 (s, 1H), 7.22 (s, 1H), 3.68 (s, 3H), 2.97 (s, 3H),
2.30 (s, 3H), 2.11 (s, 3H), 1.68 (s, 6H). ESI-MS calculated for
C.sub.27H.sub.27N.sub.4O.sub.4 [M+H].sup.+=471.20; Observed:
471.67.
##STR00407##
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)-N,2-dimethylpropanamide
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)phenyl)-2-methylpropanoic acid (Cpd No. 187, 20 mg,
0.043 mmol), EDCI-HCl (60 mg, 0.3 mmol), and HOBt-H.sub.2O (45 mg,
0.3 mmol) were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.1
mL) was added followed by addition of DMF (3 mL). Methyl amine-HCl
(14 mg, 0.2 mmol) was added and the reaction mixture was stirred
for 16 h. The reaction mixture was purified by reverse phase HPLC
affording the title compound as a salt of CF.sub.3CO.sub.2H (17 mg,
67% yield). .sup.1H NMR (MeOD-d4, 300 MHz): 7.97-7.88 (m, 2H),
7.88-7.76 (m, 2H), 7.55 (s, 1H), 7.27 (s, 1H), 3.70 (s, 3H), 2.97
(s, 3H), 2.73 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H), 1.67 (s, 6H).
ESI-MS calculated for C.sub.28H.sub.30N.sub.5O.sub.3
[M+H].sup.+=484.23; Observed: 484.42.
##STR00408##
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)-2-methyl-N-(piperidin-4-yl)propanamide
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)phenyl)-2-methylpropanoic acid (Cpd No. 187, 80 mg, 0.17
mmol), EDCI-HCl (191 mg, 1 mmol), and HOBt-H.sub.2O (135 mg, 1
mmol) were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.3 mL)
was added followed by addition of DMF (5 mL).
4-Amino-1-Boc-piperidine (80 mg, 0.4 mmol) was added and the
reaction mixture was stirred for 16 h. The reaction was quenched
with NaHCO.sub.3 saturated and the aqueous layer was extracted with
ethyl acetate. The combined organic layers were washed with 10%
citric acid aqueous solution, brine, and concentrated on a rotary
evaporator. The residue was dissolved in CH.sub.2Cl.sub.2 (4 mL)
and CF.sub.3CO.sub.2H (4 mL) was added and the mixture was stirred
for 1 h before purification on a reverse phase HPLC affording the
title compound as a salt of CF.sub.3CO.sub.2H (47.6 mg, 42% yield).
.sup.1H NMR (MeOD-d4, 300 MHz): 7.98-7.90 (m, 2H), 7.84-7.76 (m,
2H), 7.56 (s, 1H), 7.28 (s, 1H), 4.10-3.90 (m, 1H), 3.70 (s, 3H),
3.40 (dt, J=12.64, 2.99 Hz, 2H), 3.04 (td, J=13.06, 2.82 Hz, 2H),
2.97 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H), 2.80-1.94 (m, 2H),
1.86-1.70 (m, 2H), 1.69 (s, 6H). ESI-MS calculated for
C.sub.32H.sub.37N.sub.6O.sub.3 [M+H].sup.+=553.29; Observed:
553.58.
##STR00409##
2-(3-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)phenyl)-2-methyl-1-(4-morpholinopiperidine-1-yl)propan-1-one
(Cpd. No. 190)
Example of general amide condensation method promoted by EDCI-HCl:
Cpd No. 187 (40 mg, 0.1 mmol), HOBt (0.6 mmol) and EDCI-HCl (0.6
mmol) were placed in a round-bottom flask. To this flask,
EtN(i-Pr).sub.2 (0.3 mL) and anhydrous DMF (3 mL) were added.
4-Morpholinopiperidine (50 mg, 0.3 mmol) was then added in one
portion. The mixture was stirred at room temperature for 12 h
before quenching with water. The mixture was purified on reverse
phase HPLC to yield Cpd, No. 190 as a CF.sub.3CO.sub.2H salt in 11
mg (15%). .sup.1H NMR (300 MHz, MeOD-d4): 8.04-7.96 (m, 1H),
7.90-7.82 (m, 2H), 7.74-7.66 (m, 1H), 7.57 (s, 1H), 7.23 (s, 1H),
4.00-3.85 (m, 2H), 3.80-3.60 (m, 2H), 3.68 (s, 3H), 3.40-3.20 (m,
2H), 3.10-2.90 (m, 2H), 2.97 (s, 3H), 2.75-2.60 (m, 2H), 2.32 (s,
3H), 2.14 (s, 3H), 2.10-1.80 (m, 2H), 1.67 (s, 6H), 1.50-1.20 (m,
2H). ESI-MS calculated for C.sub.36H.sub.43N.sub.6O.sub.4
[M+H].sup.+=623.33; observed: 623.58.
##STR00410##
Methyl 3-bromo-5-(chloromethyl)benzoate (CE157)
Methyl 3-bromo-5-(hydroxymethyl)benzoate (2.45 g, prepared
according to literature method, JACS, 2012, v 134, 1673-1679),
pyridine (3 mL) and anhydrous THF (50 mL) were mixed in a
round-bottom flask, which was cooled with an ice-water bath.
MeSO.sub.2--Cl (1.55 mL) was added via a syringe and the reaction
was warmed up to ambient temperature for 6 h. The mixture was
quenched with water and the aqueous layer was extracted with ethyl
acetate. The organic layers were combined, dried and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield CD157 in 1.2 g
(62% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.12 (t, J=1.62 Hz,
1H), 7.98 (t, J=1.48 Hz, 1H), 7.73 (t, J=1.75 Hz, 1H), 4.57 (s,
2H), 3.93 (s, 3H).
Methyl 3-bromo-5-(cyanomethyl)benzoate (CE163)
CE157 (1.2 g) was dissolved in DMF (30 mL). NaCN (450 mg) was added
and the reaction mixture was heated at 45.degree. C. for 12 h. The
mixture was quenched with water and the aqueous layer was extracted
with ethyl acetate. The organic layers were combined, dried and the
volatile components were removed on a rotary evaporator. The
residue was purified by flash column chromatography to yield CE163
in 0.76 g (49% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.12 (s,
1H), 7.93 (s, 1H), 7.70 (s, 1H), 3.94 (s, 3H), 3.82 (s, 2H). ESI-MS
calculated for C.sub.10H.sub.9.sup.79BrNO.sub.2 [M+H].sup.+=253.98;
observed: 256.25.
Methyl 3-bromo-5-(2-cyanopropane-2-yl)benzoate (CE169)
CE163 (1.5 g) was dissolved in anhydrous DMSO (10 mL) and was
cooled with an ice-water bath. NaH (960 mg, 60% in mineral oil) was
added in small portions and the mixture was stirred for additional
20 min. MeI (1.94 mL) was added via a syringe and the mixture was
warmed up to room temperature and stirred overnight. The mixture
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined, dried and the
volatile components were removed on a rotary evaporator. The
residue was purified by flash column chromatography to yield CE169
in 1.47 g (87% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.13 (dd,
J=1.81, 1.40 Hz, 1H), 8.05 (s, J=1.82, 1.45 Hz, 1H), 7.82 (t,
J=1.87 Hz, 1H), 3.964 (s, 3H), 1.75 (s, 6H). .sup.13C NMR (75 MHz,
CDCl.sub.3): 165.40, 144.16, 133.01, 132.85, 132.36, 125.10,
123.64, 123.30, 52.86, 37.17, 29.16.
Methyl
3-(2-cyanopropan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2--
yl)benzoate (CE171)
CE169 (1.47 g, 5.2 mmol), bis(pinacolato)diboron (2.54 g, 10 mmol),
and potassium acetate (1.5 g, 15 mmol) were added to a round-bottom
flask Anhydrous 1,4-dixoane (20 mL) was added and the flask was
degassed and refilled with nitrogen. Pd(dppf)Cl.sub.2 (183 mg, 0.26
mmol) was added and the system was degassed again followed by
heating at 100.degree. C. for 16 h. The reaction mixture was cooled
to ambient temperature and diluted by CH.sub.2Cl.sub.2. The
solution was filtered through a pad of celite and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield the title compound
in 1.9 g (with impurity, >95% yield). .sup.1H NMR (300 MHz,
CDCl.sub.3): 8.32 (s, 1H), 8.14 (s, 1H), 8.01 (s, 1H), 3.84 (s,
3H), 1.69 (s, 6H), 1.27 (s, 12H). .sup.13C NMR (75 MHz,
CDCl.sub.3): 170.96, 166.51, 141.30, 135.65, 135.33, 130.32,
128.80, 124.08, 83.42, 52.19, 37.04, 29.05, 24.85. ESI-MS
calculated for C.sub.18H.sub.24BNNaO.sub.4 [M+H].sup.+=352.17;
observed: 352.42.
##STR00411##
3-(2-Cyanopropan-2-yl)-5-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-
-9H-pyrimido[4,5-b]indol-4-yl)benzoic acid (Cpd. No. 191)
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 890 mg, 2.6 mmol) and CE171 (1.9 g, 5.2 mmol),
1,2-dimethoxyethane (20 mL), and Na.sub.2CO.sub.3 (2 M, 9 mL) were
added. The system was degassed to remove oxygen and nitrogen was
refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (106 mg, 0.13 mmol)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and acidified to pH=2 followed by
extraction with ethyl acetate. The organic layers were combined and
the volatile components were removed on a rotary evaporator. The
residue was dissolved in THF (10 mL) and water (10 mL).
LiOH--H.sub.2O (420 mg, 10 mmol) was added and the solution was
stirred at ambient temperature for 24 h. The reaction mixture was
extracted with diethyl ether followed by acidification to pH=2 and
subsequent extraction with ethyl acetate. The organic layers were
combined and the volatile components were removed on a rotary
evaporator. The residue was purified by reverse phase HPLC to yield
the title compound in 328 mg (25% yield over two steps). .sup.1H
NMR (300 MHz, MeOD-d4): 8.72 (t, J=1.57 Hz, 1H), 8.59 (t, J=1.52
Hz, 1H), 8.42 (t, J=1.53 Hz, 1H), 7.57 (s, 1H), 7.32 (s, 1H), 3.71
(s, 3H), 2.98 (s, 3H), 2.30 (s, 3H), 2.11 (s, 3H), 1.89 (s, 6H).
ESI-MS calculated for C28H26N5O4 [M+H].sup.+=496.20; observed:
496.25.
##STR00412##
3-(2-Cyanopropan-2-yl)-5-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-
-9H-pyrimido[4,5-b]indol-4-yl)-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4--
yl)benzamide (Cpd. No. 192)
Example of general amide condensation method promoted by EDCI-HCl:
Cpd. No. 191 (30 mg), HOBt (84 mg, 0.6 mmol), and EDCI-HCl (120 mg,
0.6 mmol) were placed in a round-bottom flask. To this flask,
EtN(i-Pr).sub.2 (0.3 mL) and anhydrous DMF (3 mL) were added.
1-(Tetrahydro-2H-pyran-4-yl)-4-piperidinamine dihydrochloride (60
mg, 0.3 mmol) was then added in one portion. The mixture was
stirred at room temperature for 12 h before quenching with water.
The mixture was purified on reverse phase HPLC to yield Cpd. No.
192 as a CF.sub.3CO.sub.2H salt in 33 mg (71% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 8.57 (t, J=1.52 Hz, 1H), 8.43 (t, J=1.67 Hz,
1H), 8.36 (t, J=1.65 Hz, 1H), 7.57 (s, 1H), 7.27 (s, 1H), 4.30-4.15
(m, 1H), 4.15-4.00 (m, 2H), 3.78-3.66 (m, 2H), 3.70 (s, 3H),
3.54-3.36 (m, 3H), 3.26-3.12 (m, 2H), 2.98 (s, 3H), 2.40-2.28 (m,
2H), 2.31 (s, 3H), 2.13 (s, 3H), 2.10-1.90 (m, 4H), 1.90 (s, 6H),
1.90-1.70 (m, 2H). ESI-MS calculated for
C.sub.38H.sub.44N.sub.7O.sub.4 [M+H].sup.+=662.35; observed:
662.58.
##STR00413##
3-(2-Cyanopropan-2-yl)-5-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-
-9H-pyrimido[4,5-b]indol-4-yl)-N-(1-methylpiperidin-4-yl)benzamide
(Cpd. No. 193)
Example of general amide condensation method promoted by EDCI-HCl:
Cpd. No. 191 (30 mg, 0.05 mmol), HOBt (60 mg, 0.4 mmol), and
EDCI-HCl (80 mg, 0.4 mmol) were placed in a round-bottom flask. To
this flask, EtN(i-Pr).sub.2 (0.2 mL) and anhydrous DMF (3 mL) were
added. N-methylpiperidin-4-amine (23 mg, 0.2 mmol) was then added
in one portion. The mixture was stirred at room temperature for 12
h before quenching with water. The mixture was purified on reverse
phase HPLC to yield Cpd. No. 193 as a CF.sub.3CO.sub.2H salt in 40
mg (95% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.57 (s, 1H), 8.43
(s, 1H), 8.37 (s, 1H), 7.57 (s, 1H), 7.27 (s, 1H), 4.30-4.16 (m,
1H), 3.70 (s, 3H), 3.68-3.56 (m, 2H), 3.26-3.10 (m, 2H), 2.98 (s,
3H), 2.90 (s, 3H), 2.34-2.20 (m, 2H), 2.31 (s, 3H), 2.12 (s, 3H),
2.08-1.92 (m, 2H), 1.90 (s, 6H). ESI-MS calculated for
C.sub.34H.sub.38N.sub.7O.sub.3 [M+H].sup.+=592.30; observed:
592.58.
##STR00414##
3-(2-Cyanopropan-2-yl)-5-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-
-9H-pyrimido[4,5-b]indol-4-yl)-N-(1-(oxetan-3-yl)piperidin-4-yl)benzamide
(Cpd. No. 194)
Example of general amide condensation method promoted by EDCI-HCl:
Cpd. No. 191 (40 mg, 0.09 mmol), HOBt (70 mg, 0.5 mmol), and
EDCI-HCl (100 mg, 0.5 mmol) were placed in a round-bottom flask. To
this flask, EtN(i-Pr).sub.2 (0.3 mL) and anhydrous DMF (3 mL) were
added. 1-oxetan-3-ylpiperidin-4-amine (70 mg, 0.3 mmol) was then
added in one portion. The mixture was stirred at room temperature
for 12 h before quenching with water. The mixture was purified on
reverse phase HPLC to yield Cpd. No. 194 as a CF.sub.3CO.sub.2H
salt in 37 mg (61% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.57 (s,
1H), 8.44 (s, 1H), 8.36 (s, 1H), 7.57 (s, 1H), 7.26 (s, 1H),
4.90-4.80 (m, 4H), 4.50-4.36 (m, 1H), 4.34-4.20 (m, 1H), 3.70 (s,
3H), 3.66-3.46 (m, 2H), 3.18-2.96 (m, 2H), 2.98 (s, 3H), 2.38-2.22
(m, 2H), 2.31 (s, 3H), 2.16-1.96 (m, 2H), 2.12 (s, 3H), 1.90 (s,
6H). ESI-MS calculated for C.sub.36H.sub.40N.sub.7O.sub.4
[M+H].sup.+=634.31; observed: 634.50.
##STR00415##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-(oxetan-3-yl)piperazin-1-yl)methanone
(Cpd. No. 195)
Cpd. No. 195 was prepared from Cpd. No. 165 (40 mg) and
1-oxetan-3-yl-piperazine (45 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 195 as a CF.sub.3CO.sub.2H
salt in 23 mg (38% yield). .sup.1H NMR (300 MHz, MeOD-d4):
8.20-8.00 (m, 2H), 8.00-7.75 (m, 3H), 7.75-7.50 (m, 1H), 7.54 (s,
1H), 6.30-6.10 (m, 1H), 5.00-4.80 (m, 4H), 4.60-4.20 (m, 3H),
3.80-3.60 (m, 2H), 3.60-3.40 (m, 2H), 3.30-3.10 (m, 1H), 3.18 (s,
3H), 3.03 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS calculated
for C.sub.35H.sub.35N.sub.6O.sub.4 [M+H].sup.+=603.27; observed:
603.67.
##STR00416##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(1-methylpiperidin-4-yl)-1-naphthamide (Cpd. No. 196)
Cpd. No. 196 was prepared from Cpd. No. 165 (48 mg) and
N-methylpiperidin-4-amine (35 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 196 as a CF.sub.3CO.sub.2H
salt in 63 mg (91% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.44 (d,
J=8.48 Hz, 1H), 8.05 (d, J=7.12 Hz, 1H), 7.95 (d, J=7.33 Hz, 1H),
7.85-7.73 (m, 2H), 7.66-7.58 (m, 1H), 7.54 (s, 1H), 6.15 (s, 1H),
4.46-4.26 (m, 1H), 3.74-3.60 (m, 2H), 3.34-3.16 (m, 2H), 3.17 (s,
3H), 3.02 (s, 3H), 2.93 (s, 3H), 2.50-2.36 (m, 2H), 2.25 (s, 3H),
2.10-1.90 (m, 2H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.3 [M+H].sup.+=575.28; observed:
575.67.
##STR00417##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(1-(oxetan-3-yl)piperidin-4-yl)-1-naphthamide (Cpd. No.
197)
Cpd. No. 197 was prepared from Cpd. No. 165 (48 mg) and
1-oxetan-3-ylpiperidin-4-amine-2HCl (66 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 197 as a
CF.sub.3CO.sub.2H salt in 40 mg (54% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.44 (d, J=8.48 Hz, 1H), 8.05 (d, J=7.33 Hz, 1H), 7.97
(d, J=7.31 Hz, 1H), 7.84-7.72 (m, 2H), 7.66-7.56 (m, 1H), 7.55 (s,
1H), 6.15 (s, 1H), 4.94-4.84 (m, 4H), 4.56-4.32 (m, 2H), 3.70-3.50
(m, 2H), 3.26-3.10 (m, 2H), 3.17 (s, 3H), 3.02 (s, 3H), 2.50-2.36
(m, 2H), 2.25 (s, 3H), 2.20-2.00 (m, 2H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.36H.sub.37N.sub.6O.sub.4 [M+H].sup.+=617.29;
observed: 617.92.
##STR00418##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-(methylsulfonyl)piperazin-1-yl)methanone
(Cpd. No. 198)
Cpd No. 198 was prepared from Cpd. No. 165 (48 mg) and
1-methanesulfonyl-piperazine hydrochloride (48 mg) using general
amide condensation method promoted by EDCI-HCl. The reaction
mixture was purified by reverse phase HPLC to yield Cpd. No. 198 as
a CF.sub.3CO.sub.2H salt in 54 mg (73% yield). .sup.1H NMR (300
MHz, MeOD-d.sub.4): 8.18-8.04 (m, 2H), 7.94-7.76 (m, 3H), 7.72-7.60
(m, 1H), 7.56-7.52 (m, 1H), 6.24-6.20 (m, 1H), 4.20-4.00 (m, 2H),
3.60-3.38 (m, 4H), 3.30-3.10 (m, 2H), 3.20 (s, 3H), 3.02 (s, 3H),
2.91 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for
C.sub.33H.sub.33N.sub.6O.sub.5S [M+H].sup.+=625.22; observed:
625.80.
##STR00419##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-hydroxyethyl)-1-naphthamide (Cpd No. 199)
Cpd. No. 199 was prepared from Cpd. No. 165 (47 mg) and
2-aminoethanol (20 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 199 as a CF.sub.3CO.sub.2H salt in 22
mg (35% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.49 (d, J=8.46 Hz,
1H), 8.04 (d, J=7.29 Hz, 1H), 7.98 (d, J=7.32 Hz, 1H), 7.84-7.74
(m, 2H), 7.67-7.58 (m, 1H), 7.54 (s, 1H), 6.18 (s, 1H), 3.90-3.84
(m, 2H), 3.73-3.63 (m, 2H), 3.19 (s, 3H), 3.02 (s, 3H), 2.26 (s,
3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.30H.sub.28N.sub.5O.sub.4 [M+H].sup.+=522.21; observed:
522.50.
##STR00420##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-methoxyethyl)-1-naphthamide (Cpd. No. 200)
Cpd. No. 200 was prepared from Cpd. No. 165 (40 mg) and
2-methoxyethylamine (24 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 200 as a CF.sub.3CO.sub.2H salt in 32
mg (58% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.47 (d, J=8.52 Hz,
1H), 8.05 (d, J=8.52 Hz, 1H), 7.94 (d, J=7.33 Hz, 1H), 7.84-7.72
(m, 2H), 7.68-7.58 (m, 1H), 7.54 (s, 1H), 6.17 (s, 1H), 3.78-3.65
(m, 4H), 3.46 (s, 3H), 3.19 (s, 3H), 3.02 (s, 3H), 2.25 (s, 3H),
2.06 (s, 3H). ESI-MS calculated for C.sub.31H.sub.30N.sub.5O.sub.4
[M+H].sup.+=536.23; observed: 536.25.
##STR00421##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(1-isopropylpiperidin-4-yl)-1-naphthamide (Cpd. No.
201)
Cpd. No. 201 was prepared from Cpd. No. 165 (40 mg) and
N-isopropylpiperidin-4-amine (42 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 201 as a
CF.sub.3CO.sub.2H salt in 22 mg (30% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 9.18 (d, J=7.22 Hz, NH, 1H), 8.44 (d, J=8.54 Hz, 1H),
8.04 (d, J=7.30 Hz, 1H), 7.95 (d, J=7.30 Hz, 1H), 7.85-7.73 (m,
2H), 7.68-7.58 (m, 1H), 7.54 (s, 1H), 6.15 (s, 1H), 4.46-4.28 (m,
1H), 3.70-3.52 (m, 3H), 3.50-3.20 (m, 2H), 3.17 (s, 3H), 3.02 (s,
3H), 2.56-2.30 (m, 2H), 2.26 (s, 3H), 2.14-1.96 (m, 2H), 2.06 (s,
3H). ESI-MS calculated for C.sub.36H.sub.39N.sub.6O.sub.3
[M+H].sup.+=603.31; observed: 603.75.
##STR00422##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-(2-methoxyethoxy)ethyl)-1-naphthamide (Cpd. No.
202)
Cpd. No. 202 was prepared from Cpd. No. 165 (40 mg) and
2-(2-methoxyethoxy)ethanamine (40 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 202 as a
CF.sub.3CO.sub.2H salt in 27 mg (47% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.48 (d, J=8.50 Hz, 1H), 8.05 (d, J=7.33 Hz, 1H), 7.96
(d, J=7.33 Hz, 1H), 7.84-7.73 (m, 2H), 7.66-7.58 (m, 1H), 7.54 (s,
1H), 6.17 (s, 1H), 3.82-3.68 (m, 6H), 3.64-3.56 (m, 2H), 3.36 (s,
3H), 3.19 (s, 3H), 3.02 (s, 3H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.33H.sub.34N.sub.5O.sub.5 [M+H].sup.+=580.26;
observed: 580.58.
##STR00423##
N-(2-Cyanoethyl)-4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-py-
rimido[4,5-b]indol-4-yl)-1-naphthamide (Cpd. No. 203)
Cpd. No. 203 was prepared from Cpd. No. 165 (40 mg) and
aminoacetonitrile (21 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 203 as a CF.sub.3CO.sub.2H salt in 14
mg (25% yield). ESI-MS calculated for
C.sub.31H.sub.27N.sub.6O.sub.3 [M+H].sup.+=531.21; observed:
531.42.
##STR00424##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-(tetrahydro-2H-pyran-4-yl)piperazin-1-yl)methan-
one (Cpd. No. 204)
Cpd. No. 204 was prepared from Cpd. No. 165 (40 mg) and
1-(tetrahydro-2H-pyran-4-yl)piperazine (72 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 204 as a
CF.sub.3CO.sub.2H salt in 50 mg (79% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.20-8.06 (m, 2H), 8.00-7.90 (m, 1H), 7.90-7.76 (m, 2H),
7.72-7.60 (m, 1H), 7.54 (s, 1H), 6.28-6.14 (m, 1H), 4.18-4.02 (m,
2H), 3.80-3.30 (m, 9H), 3.30 (s, 3H), 3.19 (s, 3H), 3.02 (s, 3H),
2.26 (s, 3H), 2.16-2.00 (m, 2H), 2.06 (s, 3H), 1.90-1.70 (m, 2H),
1.44-1.30 (m, 2H). ESI-MS calculated for
C.sub.37H.sub.39N.sub.6O.sub.4 [M+H].sup.+=631.30; observed:
631.37.
##STR00425##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-((1-methylpiperidin-4-yl)methyl)-1-naphthamide (Cpd. No.
205)
Cpd. No. 205 was prepared from Cpd. No. 165 (40 mg) and
(1-methylpiperidin-4-yl)methanamine (39 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 205 as a
CF.sub.3CO.sub.2H salt in 33 mg (56% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.44 (d, J=8.50 Hz, 1H), 8.05 (d, J=7.32 Hz, 1H), 7.98
(d, J=7.33 Hz, 1H), 7.85-7.73 (m, 2H), 7.67-7.58 (m, 1H), 7.54 (s,
1H), 6.15 (s, 1H), 3.68-3.48 (m, 4H), 3.18 (s, 3H), 3.14-3.00 (m,
2H), 3.02 (s, 3H), 2.90 (s, 3H), 2.25 (s, 3H), 2.20-2.20 (m, 3H),
2.06 (s, 3H), 1.76-1.60 (m, 2H). ESI-MS calculated for
C.sub.35H.sub.37N.sub.6O.sub.3 [M+H].sup.+=589.29; observed:
589.67.
##STR00426##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(2-(methylsulfonyl)ethyl)-1-naphthamide (Cpd. No.
206)
Cpd. No. 206 was prepared from Cpd. No. 165 (40 mg) and
2-aminoethylmethyl sulfone (48 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 206 as a CF.sub.3CO.sub.2H
salt in 25 mg (42% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.53 (d,
J=8.49 Hz, 1H), 8.04 (d, J=7.35 Hz, 1H), 8.01 (d, J=7.35 Hz, 1H),
7.86-7.76 (m, 2H), 7.67-7.58 (m, 1H), 7.53 (s, 1H), 6.14 (s, 1H),
4.10-4.00 (m, 2H), 3.64-3.56 (m, 2H), 3.18 (s, 3H), 3.12, 3.01 (s,
3H), 2.26 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.31H.sub.30N.sub.5O.sub.5S [M+H].sup.+=584.20; observed:
584.50.
##STR00427##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-methylpiperazin-1-yl)methanone (Cpd. No.
207)
Cpd. No. 207 was prepared from Cpd. No. 165 (40 mg) and
1-methylpiperazine (30 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 207 as a CF.sub.3CO.sub.2H salt in 50
mg (86% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.30-8.05 (m, 2H),
8.00-7.80 (m, 3H), 7.70-7.60 (m, 1H), 7.54 (s, 1H), 6.26-6.14 (m,
1H), 3.80-3.50 (m, 4H), 3.50-3.00 (m, 4H), 3.19 (s, 3H), 3.03 (s,
3H), 2.99 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated
for C.sub.33H.sub.33N.sub.6O.sub.3 [M+H].sup.+=561.26; observed:
561.50.
##STR00428##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-isopropylpiperazin-1-yl)methanone (Cpd.
No. 208)
Cpd. No. 208 was prepared from Cpd. No. 165 (40 mg) and
1-isopropylpiperazine (40 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 208 as a CF.sub.3CO.sub.2H
salt in 43 mg (73% yield). .sup.1H NMR (300 MHz, MeOD-d4):
8.30-8.00 (m, 2H), 8.00-7.75 (m, 3H), 7.75-7.60 (m, 1H), 7.60-7.50
(m, 1H), 6.20-6.10 (m, 1H), 3.80-3.00 (m, 9H), 3.19 (s, 3H), 3.02
(s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). 1.42 (d, J=6.60 Hz, 6H).
ESI-MS calculated for C.sub.35H.sub.37N.sub.6O.sub.3
[M+H].sup.+=589.29; Observed: 589.50.
##STR00429##
Methyl 5-isopropyl-1H-pyrrole-2-carboxylate (CE156)
Step 1: Isobutyl aldehyde (2.2 g, 30 mmol), L-glycine methyl ester
(3.45 g, 30 mmol) and NaHCO.sub.3 (3.36 g, 40 mmol) were mixture in
a round-bottom flask followed by addition of anhydrous THF (100
mL). The reaction mixture was stirred at room temperature for
overnight. The solid was filter off and the solution was
concentrated on a rotary evaporator. The major of remaining residue
(3.65 g) was imine and was used without further purification.
Step 2: Ph.sub.3P (340 mg, 1.3 mmol) and Cu(MeCN).sub.4PF.sub.6
(483 mg, 1.3 mmol) were added to a dry round-bottom flask.
Anhydrous THF (100 mL) was added followed by addition of Et.sub.3N
(2.1 mL, 11.7 mmol). The imine obtained from step 1 (3.65 g, 25.5
mmol) was added as a THF solution and
trans-1,2-Bis(phenylsulfonyl)ethylene (8.0 g, 26 mmol) was added in
small portions. The system was degassed and refilled with nitrogen.
The reaction mixture was stirred at room temperature overnight. DBU
(7.8 mL, 52 mmol) was then added via s syringe and the mixture was
stirred at room temperature for 4 h. The mixture was diluted with
ethyl acetate and wash with 1N HCl to remove DBU. The organic layer
was dried, concentrated on a rotary evaporator. The remaining
residue was purified by flash column chromatography to yield CE156
in 1.52 g (35% yield). The method was previously reported by Angew.
Chem. Int. Ed. 2007, 46, 9261-9264 and Chem. Eur. J. 2010, 16,
9864-9873. .sup.1H NMR (300 MHz, CDCl.sub.3): 10.43 (s, 1H), 6.91
(s, 1H), 6.03 (s, 1H), 3.89 (s, 3H), 3.14-2.96 (m, 1H), 1.35 (d,
J=6.87 Hz, 6H). .sup.13C NMR (75 MHz, CDCl.sub.3): 162.47, 145.82,
120.67, 116.07, 105.81, 51.24, 27.46, 22.38. ESI-MS calculated for
C.sub.9H.sub.14NO.sub.2 [M+H].sup.+=168.10; observed: 168.33.
Methyl 4-bromo-5-isopropyl-1H-pyrrole-2-carboxylate
CE156 (1.52 g, 9.1 mmol) was dissolved in CH.sub.2Cl.sub.2 (20 mL)
and cooled with an ice-water bath. NBS (1.62 g, 9.1 mmol) was added
in small portions and the mixture was stirred at room temperature
for 1 h. The volatile components were removed on a rotary
evaporator and the residue was used without further purification.
.sup.1H NMR (300 MHz, CDCl.sub.3): 6.83 (d, J=2.69 Hz, 1H), 3.83
(s, 3H), 3.24-3.06 (m, 1H), 1.27 (d, J=7.06 Hz, 6H).
1-tert-Butyl 2-methyl
4-bromo-5-isopropyl-1H-pyrrole-1,2-dicarboxylate (CE158)
Methyl 4-bromo-5-isopropyl-1H-pyrrole-2-carboxylate (1.52 g,
previous crude) and Boc.sub.2O (2.94 g, 15 mmol) were dissolved in
anhydrous THF (20 mL). DMAP (1.1 g, 9 mmol) was added in small
portions. The reaction was stirred at room temperature for
overnight. The volatile components were removed on a rotary
evaporator and the residue was purified by flash column
chromatography to yield CE158 in 2.65 g (84% yield over two steps)
.sup.1H NMR (300 MHz, CDCl.sub.3): 6.81 (s, 1H), 3.80 (s, 3H),
3.32-3.16 (m, 1H), 1.59 (s, 9H), 1.38 (d, J=7.16 Hz, 6H).
1-tert-Butyl 2-methyl
5-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1,-
2-dicarboxylate (CE160)
CE158 (2.65, 7.66 mmol) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.29 g, 12.3
mmol) were dissolved in anhydrous THF (20 mL). The solution was
cooled to -78.degree. C. for 15 min before BuLi (4.92 mL, 2.5 M in
THF, 12.3 mmol) was added via a syringe. The reaction was stirred
at -78.degree. C. for 6 h before quenching with saturated
NH.sub.4Cl aqueous solution. The aqueous layer was extracted with
ethyl acetate and the combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and concentrated on a
rotary evaporator. The remaining residue was purified by flash
column chromatography to yield the title compound in 1.35 g (45%
yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.16 (s, 1H), 3.75 (s,
3H), 3.35-3.15 (m, 1H), 1.58 (s, 9H), 1.37 (d, J=7.05 Hz, 6H), 1.26
(s, 12H). .sup.13C NMR (75 MHz, CDCl.sub.3): 160.62, 153.65,
150.51, 125.56, 122.24, 85.45, 83.25, 51.40, 27.73, 27.46, 24.84,
21.75. ESI-MS calculated for C.sub.20H.sub.32BNNaO.sub.6
[M+Na].sup.+=416.22; observed: 416.17.
##STR00430##
Methyl
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-
-b]indol-4-yl)-5-isopropyl-1H-pyrrole-2-carboxylate (Cpd. No.
209)
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 342 mg, 0.8 mmol), CE160 (632 mg, 1.61 mmol),
1,2-dimethoxyethane (10 mL), and Na.sub.2CO.sub.3 (2 M, 4 mL) were
added. The system was degassed to remove oxygen and nitrogen was
refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (70 mg, 0.08 mmol) was
added and the system was degassed and refilled with nitrogen. The
reaction mixture was heated at reflux for 16 h. The aqueous layer
was extracted with ethyl acetate and the organic layers were
combined and the volatile components were removed on a rotary
evaporator. The residue was purified by flash column chromatography
to yield the title compound in 140 mg (40% yield). .sup.1H NMR (300
MHz, MeOD-d4): 7.54 (s, 1H), 7.29 (s, 1H), 7.24 (s, 1H), 3.89 (s,
3H), 3.71 (s, 3H), 3.20-3.04 (m, 1H), 2.92 (s, 3H), 2.31 (s, 3H),
2.13 (s, 3H), 1.28 (d, J=6.88 Hz, 6H). ESI-MS calculated for
C.sub.26H.sub.28N.sub.5O.sub.4 [M+H].sup.+=474.21; observed:
474.42.
##STR00431##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-1H-pyrrole-2-carboxylic acid (Cpd. No. 210)
Cpd. No. 209 (140 mg, 0.30 mmol) was dissolved in THF--H.sub.2O (10
mL, 3:2). LiOH--H.sub.2O (120 mg) was added in one portion and the
mixture was stirred at ambient temperature for overnight. The
volatile components were removed on a rotary evaporator and the
remaining residues were purified by reverse phase HPLC to yield the
title compound as a salt of CF.sub.3CO.sub.2H in 40 mg (23% yield).
.sup.1H NMR (300 MHz, MeOD-d4): 7.55 (s, 1H), 7.29 (s, 1H), 7.28
(s, 1H), 3.73 (s, 3H), 3.20-3.04 (m, 1H), 2.93 (s, 3H), 2.31 (s,
3H), 2.13 (s, 3H), 1.29 (d, J=6.97 Hz, 6H). ESI-MS calculated for
C.sub.25H.sub.26N.sub.5O.sub.4 [M+H].sup.+=460.20; observed:
460.50.
##STR00432##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-N-(1-methylpiperidin-4-yl)-1H-pyrrole-2-carboxamide
(Cpd. No. 211)
Cpd. No. 211 was prepared from Cpd. No. 210 (15 mg) and
N-methylpiperidin-4-amine (40 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 211 as a CF.sub.3CO.sub.2H
salt in 11 mg (53% yield). .sup.1H NMR (300 MHz, MeOD-d.sub.4):
7.54 (s, 1H), 7.29 (s, 1H), 7.28 (s, 1H), 4.26-4.08 (m, 1H), 3.72
(s, 3H), 3.68-3.52 (m, 2H), 3.24-3.10 (m, 3H), 2.93 (s, 3H), 2.89
(s, 3H), 2.31 (s, 3H), 2.30-2.16 (m, 2H), 2.13 (s, 3H), 1.98-1.80
(m, 2H), 1.29 (d, J=6.96 Hz, 6H). ESI-MS calculated for
C.sub.31H.sub.38N.sub.7O.sub.3 [M+H].sup.+=556.30; observed:
556.42.
##STR00433##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)-1H-pyrr-
ole-2-carboxamide (Cpd. No. 212)
Cpd. No. 212 was prepared from Cpd. No. 210 (40 mg) and
1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine dihydrochloride (54
mg) using general amide condensation method promoted by EDCI-HCl.
The reaction mixture was purified by reverse phase HPLC to yield
Cpd. No. 212 as a CF.sub.3CO.sub.2H salt in 47 mg (73% yield).
.sup.1H NMR (300 MHz, MeOD-d4): 7.54 (s, 1H), 7.31 (s, 1H), 7.29
(s, 1H), 4.26-4.12 (m, 1H), 4.12-4.02 (m, 2H), 3.76-3.64 (m, 2H),
3.72 (s, 13H), 3.52-3.38 (m, 3H), 3.26-3.10 (m, 3H), 2.93 (s, 3H),
2.34-2.22 (m, 2H), 2.31 (s, 3H), 2.13 (s, 3H), 2.10-1.70 (m, 6H),
1.29 (d, J=6.97 Hz, 6H). ESI-MS calculated for
C.sub.35H.sub.44N.sub.7O.sub.4 [M+H].sup.+=626.35; observed:
626.67.
##STR00434##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-N-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrrole-2-carboxa-
mide (Cpd. No. 213)
Cpd. No. 213 was prepared from Cpd. No. 210 (40 mg) and
1-oxetan-3-ylpiperidin-4-amine (48 mg) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 213 as a
CF.sub.3CO.sub.2H salt in 42 mg (68% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 7.55 (s, 1H), 7.34 (s, 1H), 7.30 (s, 1H), 4.90-4.80 (m,
4H), 4.50-4.40 (m, 1H), 4.30-4.14 (m, 1H), 3.72 (s, 3H), 3.66-3.50
(m, 2H), 3.26-2.94 (m, 3H), 2.93 (s, 3H), 2.38-2.20 (m, 2H), 2.30
(s, 3H), 2.18-1.90 (m, 2H), 2.13 (s, 3H), 1.29 (d, J=6.95 Hz, 6H).
ESI-MS calculated for C.sub.33H.sub.40N.sub.7O.sub.4
[M+H].sup.+=598.31; observed: 598.42.
##STR00435##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-ethyl-5-isopropyl-1H-pyrrole-2-carboxamide (Cpd. No.
214)
Cpd. No. 214 was prepared from Cpd. No. 210 (46 mg) and ethylamine
(2 M in THF, 0.3 mL) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 214 as a CF.sub.3CO.sub.2H salt in 35
mg (58% yield). .sup.1H NMR (300 MHz, MeOD-d.sub.4): 7.54 (s, 1H),
7.31 (s, 1H), 7.21 (s, 1H), 3.73 (s, 3H), 3.41 (q, J=7.25 Hz, 2H),
3.22-3.08 (m, 1H), 2.93 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H), 1.29
(d, J=6.97 Hz, 6H), 1.22 (t, J=7.24 Hz, 3H). ESI-MS calculated for
C.sub.27H.sub.31N.sub.6O.sub.3 [M+H].sup.+=487.25; observed:
487.25.
##STR00436##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-5-isopropyl-1H-pyrrol-2-yl)(4-isopropylpiperazin-1-yl)methanone
(Cpd. No. 215)
Cpd. No. 215 was prepared from Cpd. No. 210 (46 mg) and
1-isopropylpiperazine (40 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 215 as a CF.sub.3CO.sub.2H
salt in 40 mg (59% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.55 (s,
1H), 7.20 (s, 1H), 7.05 (s, 1H), 3.80-3.00 (m, 9H), 3.10 (septet,
J=7.02 Hz, 1H), 3.72 (s, 3H), 2.94 (s, 3H), 2.31 (s, 3H), 2.13 (s,
3H), 1.40 (d, J=6.57 Hz, 6H), 1.28 (d, J=6.90 Hz, 6H). ESI-MS
calculated for C.sub.32H.sub.40N.sub.7O.sub.3 [M+H].sup.+=570.32;
Observed: 570.58.
##STR00437##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-5-isopropyl-1H-pyrrol-2-yl)(4-methylpiperazin-1-yl)methanone
(Cpd. No. 216)
Cpd. No. 216 was prepared from Cpd. No. 210 (40 mg) and
1-methylpiperazine (30 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 216 as a CF.sub.3CO.sub.2H salt in 24
mg (43% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.55 (s, 3H), 7.20
(s, 1H), 7.03 (s, 1H), 3.72 (s, 3H), 3.70-3.00 (m, 8H), 3.10
(septet, J=6.96 Hz, 1H), 2.96 (s, 3H), 2.94 (s, 3H), 2.31 (s, 3H),
2.13 (s, 3H), 1.28 (d, J=6.97 Hz, 6H). ESI-MS calculated for
C.sub.30H.sub.36N.sub.7O.sub.3 [M+H].sup.+=542.29; Observed:
542.42
##STR00438##
tert-Butyldimethyl((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphtha-
len-1-yl)methoxy)silane (CE187)
Step 1: (4-Bromonaphthalen-1-yl)methanol (10.06 g, 43 mmol) and
TBS-Cl (8.46 g, 56 mmol) were dissolved in anhydrous THF (100 mL)
and the mixture was cooled with an ice-water bath. Imidazole (4.42
g, 65 mmol) was added in small portions and the reaction was warmed
up to ambient temperature overnight. Water was added and the
aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous sodium
sulfate, and concentrated on a rotary evaporator. The remaining
residue was purified by flash column chromatography to yield
((4-bromonaphthalen-1-yl)methoxy)(tert-butyl)dimethylsilane in
13.82 g (91% yield).
Step 2: ((4-bromonaphthalen-1-yl)methoxy)(tert-butyl)dimethylsilane
(13.82 g, 39.4 mmol) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (11.16 g, 60
mmol) were dissolved in anhydrous THF (100 mL). The solution was
cooled to -78.degree. C. for 15 min before BuLi (24 mL, 2.5 M in
THF, 60 mmol) was added via a syringe. The reaction was stirred at
-78.degree. C. for 6 h before quenching with saturated NH.sub.4Cl
aqueous solution. The aqueous layer was extracted with ethyl
acetate and the combined organic layers were washed with brine,
dried over anhydrous sodium sulfate, and concentrated on a rotary
evaporator. The remaining residue was purified by flash column
chromatography to yield the title compound in 12.46 g (79% yield).
.sup.1H NMR (300 MHz, CDCl.sub.3): 8.80 (d, J=7.61 Hz, 1H), 8.07
(d, J=7.07 Hz, 1H), 7.96 (dd, J=7.89, 1.31 Hz, 1H), 7.60 d, J=7.11
Hz, 1H), 7.57-7.45 (m, 2H), 7.26 (s, 1H), 5.22 (s, 2H), 1.42 (s,
12H), 0.95 (s, 9H), 0.11 (s, 6H).
##STR00439##
4-(4-(4-(((tert-Butyldimethylsilyl)oxy)methyl)naphthalen-1-yl)-6-methoxy-2-
-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
(CE191)
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.47 g, 16 mmol), CE187 (12.46 g, 31.3 mmol),
1,2-dimethoxyethane (100 mL), and Na.sub.2CO.sub.3 (2 M, 50 mL)
were added. The system was degassed to remove oxygen and nitrogen
was refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.30 mg, 1.6
mmol) was added and the system was degassed and refilled with
nitrogen. The reaction mixture was heated at reflux for 16 h. The
aqueous layer was extracted with ethyl acetate and the organic
layers were combined and dried The volatile components were removed
on a rotary evaporator. The remaining residue was purified by flash
column chromatography to yield the title compound in 3.86 g (41%
yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.23 (d, J=8.45 Hz, 1H),
7.77 (d, J=7.28 Hz, 1H), 7.72-7.60 (m, 2H), 7.58-7.48 (m, 1H),
7.38-7.32 (m, 1H), 7.30 (s, 1H), 6.15 (s, 1H), 5.30 (q, J=13.39 Hz,
2H), 3.09 (s, 3H), 2.83 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H), 0.92
(s, 9H), 0.13 (s, 6H).
##STR00440##
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)methanol (CE192)
CE191 (1.9 g, 3.3 mmol) was dissolved in THF (20 mL) and TBAF (6
mL, 1.0 M in THF, 6 mmol) was added via a syringe. The reaction
mixture was stirred at ambient temperature for overnight. The
volatile components were removed on a rotary evaporator. The
remaining residue was purified by flash column chromatography to
yield the title compound in 1.2 g (78% yield). Further purification
on reverse phase HPLC afforded the CE192 as a CF.sub.3CO.sub.2H
salt. .sup.1H NMR (300 MHz, MeOD-d4): 8.39 (d, J=8.50 Hz, 1H),
8.046-7.94 (m, 2H), 7.86-7.70 (m, 2H), 7.64-7.56 (m, 1H), 7.53 (s,
1H), 6.18 (s, 1H), 5.34 (d, J=14.15 Hz, 1H), 5.28 (d, J=14.11 Hz,
1H), 3.16 (s, 3H), 3.01 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.28H.sub.25N.sub.4O.sub.3 [M+H].sup.+=465.19;
observed: 465.32.
##STR00441##
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-1-naphthaldehyde (CE194)
CE192 (1.2 g, 2.6 mmol) was dissolved in DMSO (20 mL). IBX (1.46 g,
5.2 mmol) was added in small portions and the mixture was stirred
at room temperature for overnight before quenching with
NaHCO.sub.3. The aqueous layer was extracted with ethyl acetate and
the organic layers were combined and dried. The volatile components
were removed on a rotary evaporator. The remaining residue was
purified by flash column chromatography to yield the title compound
in 1.19 g (98% yield). .sup.1H NMR (300 MHz, DMSO-d6): 12.29 (s,
1H), 10.56 (s, 1H), 9.34 (d, J=8.65 Hz, 1H), 8.42 (d, J=7.33 Hz,
1H), 8.01 (d, J=7.24 Hz, 1H), 7.85-7.75 (m, 2H), 7.60-7.53 (m, 1H),
7.33 (s, 1H), 6.12 (s, 1H), 3.11 (s, 3H), 2.79 (s, 3H), 2.21 (s,
3H), 1.99 (s, 3H). ESI-MS calculated for
C.sub.28H.sub.23N.sub.4O.sub.3 [M+H].sup.+=463.18; observed:
463.67.
##STR00442##
4-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]-
indol-4-yl)naphthalen-1-yl)methyl)morpholine (Cpd. No. 217)
CE194 (50 mg), morpholine (18 mg, 0.2 mmol), and acetic acid (0.1
mL) were dissolved in anhydrous THF (3 mL). NaBH(OAc).sub.3 (120
mg, 0.5 mmol) was added in one portion and the mixture was stirred
at ambient temperature for overnight. The volatile components were
removed on a rotary evaporator. The remaining residue was purified
by reverse phase HPLC to yield the title compound Cpd. No. 217 as a
CF.sub.3CO.sub.2H salt in 31 mg (47% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.61 (d, J=8.55 Hz, 1H), 8.15 (d, J=7.41 Hz, 1H), 8.08
(d, J=7.41 Hz, 1H), 7.92-7.84 (m, 2H), 7.72-7.64 (m, 1H), 7.54 (s,
1H), 6.18 (s, 1H), 5.15 (d, J=13.59 Hz, 1H), 5.07 (d, J=13.59 Hz,
1H), 4.10-3.80 (m, 4H), 3.54-3.46 (m, 4H), 3.17 (s, 3H), 3.02 (s,
3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.32N.sub.5O.sub.3 [M+H].sup.+=534.25; observed:
557.50.
##STR00443##
4-(6-Methoxy-2-methyl-4-(4-((4-methylpiperazin-1-yl)methyl)naphthalen-1-yl-
)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (Cpd. No.
218)
CE194 (50 mg), 1-methylpiperazine (20 mg, 0.2 mmol), and acetic
acid (0.1 mL) were dissolved in anhydrous THF (3 mL).
NaBH(OAc).sub.3 (120 mg, 0.5 mmol) was added in one portion and the
mixture was stirred at ambient temperature for overnight. The
volatile components were removed on a rotary evaporator. The
remaining residue was purified by reverse phase HPLC to yield the
title compound Cpd. No. 218 as a CF.sub.3CO.sub.2H salt in 19 mg
(29% yield). .sup.1H NMR (300 MHz, MeOD-d.sub.4): 8.62 (d, J=8.35
Hz, 1H), 7.97 (d, J=7.29 Hz, 1H), 7.90 (d, J=7.36 Hz, 1H),
7.80-7.72 (m, 2H), 7.64-7.56 (m, 1H), 7.53 (s, 1H), 6.16 (s, 1H),
4.35 (s, 2H), 3.48-3.30 (m, 4H), 3.15 (s, 3H), 3.10-2.90 (m, 4H),
3.01 (s, 3H), 2.91 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS
calculated for C.sub.33H.sub.35N.sub.6O.sub.2 [M+H].sup.+=547.28;
observed: 547.33.
##STR00444##
4-(6-Methoxy-2-methyl-4-(4-(piperazin-1-ylmethyl)naphthalen-1-yl)-9H-pyrim-
ido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (Cpd. No. 219)
Step 1: CE194 (300 mg, 0.7 mmol), 1-Boc-piperazine (260 mg, 1.4
mmol), and acetic acid (0.2 mL) were dissolved in anhydrous THF (5
mL). NaBH(OAc).sub.3 (445 mg, 2.1 mmol) was added in one portion
and the mixture was stirred at ambient temperature for overnight.
The volatile components were removed on a rotary evaporator. The
remaining residue was directly used for next step.
Step 2: The previous residue from step 1 was mixed with
CH.sub.2Cl.sub.2 (5 mL) followed by addition of triethylsilane (0.1
mL) and CF.sub.3CO.sub.2H (5 mL). The mixture was stirred at
ambient temperature for 2 h and the volatile components were
removed on a rotary evaporator. The remaining residue was purified
by reverse phase HPLC to yield the title compound Cpd. No. 219 as a
CF.sub.3CO.sub.2H salt in 300 mg (80% yield over two steps).
.sup.1H NMR (300 MHz, MeOD-d4): 8.61 (d, J=8.25 Hz, 1H), 8.00 (d,
J=7.31 Hz, 1H), 7.96 (d, J=7.31 Hz, 1H), 7.84-7.74 (m, 2H),
7.65-7.57 (m, 1H), 7.54 (s, 1H), 6.17 (s, 1H), 4.52 (s, 2H),
3.42-3.32 (m, 4H), 3.18-3.10 (m, 4H), 3.15 (s, 3H), 3.02 (s, 3H),
2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.32H.sub.33N.sub.6O.sub.2 [M+H].sup.+=533.27; observed:
533.25.
##STR00445##
1-(4-((4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-
-b]indol-4-yl)naphthalen-1-yl)methyl)piperazin-1-yl)ethanone (Cpd.
No. 220)
Cpd. No. 219(50 mg) was dissolved in anhydrous THF (3 mL). Acetic
anhydride (0.05 mL, 0.2 mmol) was added via a syringe and the
mixture was stirred at ambient temperature for 4 h. The volatile
components were removed on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC to yield the title
compound Cpd. No. 220 as a CF.sub.3CO.sub.2H salt in 33 mg (48%
yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.59 (d, J=8.53 Hz, 1H),
8.15 (d, J=7.42 Hz, 1H), 8.09 (d, J=7.42 Hz, 1H), 7.92-7.82 (m,
2H), 7.72-7.64 (m, 1H), 7.55 (s, 1H), 6.19 (s, 1H), 5.15 (d,
J=13.58 Hz, 1H), 5.08 (d, J=13.58 Hz, 1H), 3.96-3.82 (m, 4H),
3.60-3.50 (m, 2H), 3.50-3.40 (m, 2H), 3.18 (s, 3H), 3.02 (s, 3H),
2.25 (s, 3H), 2.17 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.3 [M+H].sup.+=575.28; observed:
575.42.
##STR00446##
4-(4-(4-((4-Isopropylpiperazin-1-yl)methyl)naphthalen-1-yl)-6-methoxy-2-me-
thyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (Cpd. No.
221)
CE194 (46 mg), 1-isopropylpiperazine (60 mg, 0.3 mmol), and acetic
acid (0.1 mL) were dissolved in anhydrous THF (5 mL).
NaBH(OAc).sub.3 (110 mg, 0.5 mmol) was added in one portion and the
mixture was stirred at ambient temperature for overnight. The
volatile components were removed on a rotary evaporator. The
remaining residue was purified by reverse phase HPLC to yield the
title compound Cpd. No. 221 as a CF.sub.3CO.sub.2H salt in 55 mg
(80% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.61 (d, J=8.19 Hz,
1H), 8.02-7.90 (m, 2H), 7.82-7.72 (m, 2H), 7.65-7.56 (m, 1H), 7.54
(s, 1H), 6.17 (s, 1H), 4.49 (d, J=13.44 Hz, 1H), 4.42 (d, J=13.44
Hz, 1H), 3.70-2.70 (m, 8H), 3.55 (septet, J=6.62 Hz, 1H), 3.16 (s,
3H), 3.02 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H), 1.38 (d, J=6.65 Hz,
6H). ESI-MS calculated for C.sub.35H.sub.39N.sub.6O.sub.2
[M+H].sup.+=575.31; Observed: 575.92
##STR00447##
N-(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)naphthalen-1-yl)-2-(4-methylpiperazin-1-yl)acetamide
(Cpd. No. 222)
Following protocol similar to Method 149, reaction of
4-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-amine (Cpd. No. 150, 45 mg, 0.1 mmol)
chloroacetyl chloride (113 mg, 1 mmol), and
N-methylpiperidin-4-amine (40 mg, 0.4 mmol) afforded the title
compound. Upon treatment of CF.sub.3CO.sub.2H followed by reverse
phase HPLC purification, the title compound was isolated in 39 mg
as a CF.sub.3CO.sub.2H salt (55% over two steps). .sup.1H NMR (300
MHz, MeOD-d4): 8.35 (d, J=8.69 Hz, 1H), 8.22 (d, J=7.83 Hz, 1H),
8.06 (d, J=7.85 Hz, 1H), 7.85-7.77 (m, 2H), 7.68-7.61 (m, 1H), 7.56
(s, 1H), 6.24 (s, 1H), 3.83 (s, 2H), 3.62-3.50 (m, 4H), 3.38-3.22
(m, 4H), 3.19 (s, 3H), 3.04 (s, 3H), 3.01 (s, 3H), 2.28 (s, 3H),
2.08 (s, 3H), ESI-MS calculated for C.sub.34H.sub.36N.sub.7O.sub.3
[M+H].sup.+=590.29; observed: 590.50.
##STR00448##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-3,3-dimethylbutan-1-ol (Cpd. No. 223)
S13 (70 mg), L-tert Leucinol (44 mg), NaHCO.sub.3 (32 mg) and
anhydrous DMSO (3 mL) were heated at 150.degree. C. for 16 h. The
mixture was then purified by reverse phase HPLC to yield Cpd. No.
223 as a CF.sub.3CO.sub.2H salt in 9 mg (9% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 7.88 (s, 1H), 7.45 (s, 1H), 4.70-4.55 (m, 1H),
4.04 (dd, J=12.02, 4.75 Hz, 1H), 4.00-3.93 (m, 1H), 3.96 (s, 3H),
2.72 (s, 3H), 2.32 (s, 3H), 2.16 (s, 3H), 1.12 (s, 9H). ESI-MS
calculated for C.sub.23H.sub.30N.sub.5O.sub.3 [M+H].sup.+=424.23;
observed: 424.60.
##STR00449##
N-benzyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-amine (Cpd. No. 224)
S13 (68 mg), L-phenyl glycine (60 mg), NaHCO.sub.3 (48 mg) and
anhydrous DMSO (3 mL) were heated at 100.degree. C. for 16 h. The
mixture was then purified by reverse phase HPLC to yield Cpd. No.
224 as a CF.sub.3CO.sub.2H salt in 52 mg (50% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 8.05 (s, 1H), 7.48-7.40 (m, 2H), 7.44 (s, 1H),
7.38-7.22 (m, 3H), 5.08 (s, 2H), 3.93 (s, 3H), 2.69 (s, 3H), 2.30
(s, 3H), 2.13 (s, 3H). ESI-MS calculated for
C.sub.24H.sub.24N.sub.5O.sub.2 [M+H].sup.+=414.19; observed:
414.60.
##STR00450##
(2S)-2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b-
]indol-4-yl)amino)-2-phenylethanol (Cpd. No. 225)
S13 (68 mg), L-phenyl glycinol (56 mg), NaHCO.sub.3 (84 mg) and
anhydrous DMSO (3 mL) were heated at 100.degree. C. for 16 h. The
mixture was then purified by reverse phase HPLC to yield Cpd. No.
225 as a CF.sub.3CO.sub.2H salt in 70 mg (62% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 8.06 (s, 1H), 7.56-7.50 (m, 2H), 7.44 (s, 1H),
7.42-7.26 (m, 3H), 5.84 (dd, J=7.56, 4.87 Hz, 1H), 4.20 (dd,
J=11.38, 7.77 Hz, 1H), 4.13 (dd, J=11.38, 4.87 Hz, 1H), 3.97 (s,
3H), 2.65 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS calculated
for C.sub.25H.sub.26N.sub.5O.sub.3 [M+H].sup.+=444.20; observed:
444.67.
##STR00451##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-3-methylbutanoic acid (Cpd. No. 226)
S13 (68 mg), valine (48 mg), NaHCO.sub.3 (84 mg) and anhydrous DMSO
(3 mL) were heated at 100.degree. C. for 16 h. The mixture was then
purified by reverse phase HPLC to yield Cpd. No. 226 as a
CF.sub.3CO.sub.2H salt in 46 mg (43% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 7.89 (s, 1H), 7.45 (s, 1H), 3.97 (s, 3H), 2.69 (s, 3H),
2.60-2.46 (m, 1H), 2.33 (s, 3H), 2.15 (s, 3H), 1.17 (t, J=6.95 Hz,
6H). ESI-MS calculated for C.sub.22H.sub.26N.sub.5O.sub.4
[M+H].sup.+=424.20; observed: 424.33.
##STR00452##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-3-methyl-N-(1-methylpiperidin-4-yl)butanamide
(Cpd. No. 227)
Cpd. No. 227 was prepared from Cpd. No. 226 (30 mg) and
N-methylpiperidin-4-amine (33 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 227 as a CF.sub.3CO.sub.2H
salt in 38 mg (85% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.94 (s,
1H), 7.46 (s, 1H), 4.84 (d, J=8.65 Hz, 1H), 4.10-3.90 (m, 1H), 3.98
(s, 3H), 3.65-3.50 (m, 2H), 3.20-3.06 (m, 2H), 2.87 (s, 3H), 2.73
(s, 3H), 2.50-2.36 (m, 1H), 2.32 (s, 3H), 2.26-2.08 (m, 2H), 2.14
(s, 3H), 1.94-1.76 (m, 2H), 1.12 (t, J=6.11 Hz, 6H). ESI-MS
calculated for C.sub.28H.sub.38N.sub.7O.sub.3 [M+H].sup.+=520.30;
observed: 520.55.
##STR00453##
(2R)-2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b-
]indol-4-yl)amino)-2-phenylethanol (Cpd. No. 228)
S13 (68 mg), D-phenylglycinol (56 mg), EtN(i-Pr).sub.2 (0.2 mL) and
anhydrous DMSO (3 mL) were heated at 100.degree. C. for 16 h. The
mixture was then purified by reverse phase HPLC to yield Cpd. No.
228 as a CF.sub.3CO.sub.2H salt in 35 mg (32% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 8.06 (s, 1H), 7.57-7.50 (m, 2H), 7.44 (s, 1H),
7.43-7.25 (m, 3H), 5.83 (dd, J=7.55, 4.93 Hz, 1H), 4.20 (dd,
J=11.38, 7.74 Hz, 1H), 4.13 (dd, J=11.38, 4.88 Hz, 1H), 3.97 (s,
3H), 2.66 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS calculated
for C.sub.25H.sub.26N.sub.5O.sub.3 [M+H].sup.+=444.20; observed:
444.25.
##STR00454##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-2-(4-fluorophenyl)ethanol (Cpd. No. 229)
S13 (70 mg), (S)-2-amino-2-(4-fluorophenyl)ethanol (60 mg),
EtN(i-Pr).sub.2 (0.1 mL) and anhydrous DMSO (3 mL) were heated at
100.degree. C. for 16 h. The mixture was then purified by reverse
phase HPLC to yield Cpd. No. 229 as a CF.sub.3CO.sub.2H salt in 9
mg (8% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.06 (s, 1H),
7.60-7.54 (m, 1H), 7.44 (s, 1H), 7.15-7.07 (m, 1H), 5.82 (dd,
J=7.37, 5.08 Hz, 1H), 4.17 (dd, J=11.35, 7.64 Hz, 1H), 4.10 (dd,
J=11.35, 5.07 Hz, 1H), 3.98 (s, 3H), 2.66 (s, 3H), 2.32 (s, 3H),
2.15 (s, 3H). ESI-MS calculated for C.sub.25H.sub.25FN.sub.5O.sub.3
[M+H].sup.+=462.19; observed: 462.25.
##STR00455##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-4-methylpentanoic acid (Cpd. No. 230)
S13 (70 mg), L-Leucine (52 mg), NaHCO.sub.3 (84 mg) and anhydrous
DMSO (3 mL) were heated at 100.degree. C. for 16 h. The mixture was
then purified by reverse phase HPLC to yield Cpd. No. 230 as a
CF.sub.3CO.sub.2H salt in 60 mg (54% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.01 (s, 1H), 7.43 (s, 1H), 5.32 (dd, J=10.40, 4.58 Hz,
1H), 3.96 (s, 3H), 2.68 (s, 3H), 2.34 (s, 3H), 2.15 (s, 3H),
2.14-2.06 (m, 1H), 2.04-1.90 (m, 1H), 1.90-1.78 (m, 1H), 1.03 (t,
J=6.93 Hz, 6H). ESI-MS calculated for
C.sub.23H.sub.28N.sub.5O.sub.4 [M+H].sup.+=438.21; observed:
438.42.
##STR00456##
(2S,3S)-2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,-
5-b]indol-4-yl)amino)-3-methylpentanoic acid (Cpd. No. 231)
S13 (70 mg), L-Isoleucine (52 mg), NaHCO.sub.3 (84 mg) and
anhydrous DMSO (3 mL) were heated at 100.degree. C. for 16 h. The
mixture was then purified by reverse phase HPLC to yield Cpd. No.
231 as a CF.sub.3CO.sub.2H salt in 54 mg (49% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 7.89 (s, 1H), 7.46 (s, 1H), 5.09 (d, J=6.64 Hz,
1H), 3.97 (s, 3H), 2.72 (s, 3H), 2.36-2.24 (m, 1H), 2.33 (s, 3H),
2.16 (s, 3H), 1.86-1.70 (m, 1H), 1.56-1.36 (m, 1H), 1.12 (d, J=6.84
Hz, 3H), 1.04 (t, J=7.37 Hz, 3H). ESI-MS calculated for
C.sub.23H.sub.28N.sub.5O.sub.4 [M+H].sup.+=438.21; observed:
438.33.
##STR00457##
(3R)-3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-3-phenylpropanoic acid (Cpd. No. 232)
S13 (684 mg), (R)-3-amino-3-phenylpropionic acid (660 mg),
NaHCO.sub.3 (800 mg) and anhydrous DMSO (10 mL) were heated at
130.degree. C. for 16 h. The mixture was then purified by reverse
phase HPLC to yield Cpd. No. 232 as a CF.sub.3CO.sub.2H salt in 270
mg (23% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.00 (s, 1H),
7.60-7.50 (m, 2H), 7.45 (s, 1H), 7.40-7.20 (m, 3H), 6.12 (t, J=5.74
Hz, 1H), 3.97 (s, 3H), 3.23 (d, J=5.81 Hz, 2H), 2.67 (s, 3H), 2.32
(s, 3H), 2.15 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.26N.sub.5O.sub.4 [M+H].sup.+=472.20; observed:
4752.25.
##STR00458##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-2-phenylacetic acid (Cpd. No. 233)
Step 1: S13 (682 mg, 2.0 mmol), (S)-tert-butyl
2-amino-2-phenylacetate (1.0 g, 4.0 mmol), NaHCO.sub.3 (800 mg, 8
mmol) and anhydrous DMSO (10 mL) were heated at 130.degree. C. for
16 h. The mixture was then diluted with water and the aqueous layer
was extracted with ethyl acetate. The organic layers were combined
and dried, and the volatile components were removed on a rotary
evaporator. The remaining residue was directly used for the next
step.
Step 2: The previous residue from step 1 was mixed with
CF.sub.3CO.sub.2H (10 mL) and water (0.5 mL) followed by addition
of triethylsilane (1 mL). The mixture was stirred at ambient
temperature for overnight and the volatile components were removed
on a rotary evaporator. The remaining residue was purified by
reverse phase HPLC to yield the title compound Cpd. No. 233 as a
CF.sub.3CO.sub.2H salt in 87 mg (8% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 7.95 (s, 1H), 7.66-7.60 (m, 2H), 7.46 (s, 1H), 7.45-7.33
(m, 3H), 6.23 (s, 1H), 3.95 (s, 3H), 2.69 (s, 3H), 2.32 (s, 3H),
2.15 (s, 3H). ESI-MS calculated for C.sub.25H.sub.24N.sub.5O.sub.4
[M+H].sup.+=458.18; observed: 458.25.
##STR00459##
3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)amino)-3-(2-fluorophenyl)propanoic acid (Cpd. No. 234)
S13 (342 mg), (R)-3-amino-3-(2-fluoro-phenyl)-propionic acid (360
mg), NaHCO.sub.3 (300 mg) and anhydrous DMSO (10 mL) were heated at
130.degree. C. for 16 h. The mixture was then purified by reverse
phase HPLC to yield Cpd. No. 234 as a CF.sub.3CO.sub.2H salt in 236
mg (39% yield). .sup.1H NMR (300 MHz, MeOD-d4): racemic, 8.01 (s,
1H), 7.56-7.48 (m, 1H), 7.10-7.30 (m, 1H), 7.46 (s, 1H), 7.22-7.10
(m, 2H), 6.31 (t, J=5.93 Hz, 1H), 3.99 (s, 3H), 3.25-3.18 (m, 2H),
2.67 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.25FN.sub.5O.sub.4 [M+H].sup.+=490.19; observed:
490.62.
##STR00460##
3-(2-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-
-pyrimido[4,5-b]indol-4-yl)amino)propanoic acid (Cpd. No. 235)
S13 (70 mg), (R)-3-amino-3-(2-chloro-phenyl)-propionic acid (80
mg), NaHCO.sub.3 (84 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for 16 h. The mixture was then purified by reverse
phase HPLC to yield Cpd. No. 235 as a CF.sub.3CO.sub.2H salt in 39
mg (31% yield). .sup.1H NMR (300 MHz, MeOD-d4): racemic, 8.04 (s,
1H), 7.62-7.54 (m, 1H), 7.50-7.46 (m, 1H), 7.46 (s, 1H), 7.34-7.24
(m, 2H), 6.37 (t, J=6.37 Hz, 1H), 4.00 (s, 3H), 3.24-3.17 (m, 2H),
2.64 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.25ClN.sub.5O.sub.4 [M+H].sup.+=506.16; observed:
506.67.
##STR00461##
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)propanoic acid (Cpd. No.
236)
S13 (68 mg), (R)-3-amino-3-(3-chlorophenyl)propanoic acid (80 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 236 as a CF.sub.3CO.sub.2H
salt in 56 mg (55% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.00 (s,
1H), 7.55 (s, 1H), 7.50-7.44 (m, 1H), 7.46 (s, 1H), 7.40-7.25 (m,
2H), 6.11 (t, J=6.09 Hz, 1H), 3.98 (s, 3H), 3.22 (d, J=6.15 Hz,
2H), 2.69 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS calculated
for C.sub.26H.sub.25.sup.35ClN.sub.5O.sub.4 [M+H].sup.+=506.16;
Observed: 506.58.
##STR00462##
3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)amino)-3-(naphthalen-1-yl)propanoic acid (Cpd. No. 237)
S13 (68 mg), 3-amino-3-(naphthalen-1-yl)propanoic acid (86 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 237 as a CF.sub.3CO.sub.2H
salt in 35 mg (28% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.31 (d,
1H, J=8.4 Hz, 1H), 8.08 (s, 1H), 7.93 (d, 1H, J=7.6 Hz, 1H), 7.83
(d, 1H, J=8.3 Hz, 1H), 7.72-7.60 (m, 2H), 7.60-7.50 (m, 1H),
7.48-7.40 (m, 1H), 7.47 (s, 1H), 6.91 (t, J=5.80 Hz, 1H), 3.98 (s,
3H), 3.36-3.28 (m, 2H), 2.57 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H).
ESI-MS calculated for C.sub.30H.sub.28N.sub.5O.sub.4
[M+H].sup.+=522.21; Observed: 522.33.
##STR00463##
2-(2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)-1,2,3,4-tetrahydroisoquinolin-1-yl)acetic acid (Cpd. No.
238)
S13 (68 mg), 2-(1,2,3,4-tetrahydroisoquinolin-1-yl)acetic acid (80
mg), NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 238 as a CF.sub.3CO.sub.2H
salt in 55 mg (45% yield). .sup.1H NMR (300 MHz, MeOD-d4):
7.48-7.40 (m, 1H), 7.46 (s, 1H), 7.35-7.20 (m, 3H), 7.29 (s, 1H),
6.40-6.24 (m, 1H), 4.67 (dd, J=13.41, 5.22 Hz, 1H), 4.14 (td,
J=12.65, 4.06 Hz, 1H), 3.70 (s, 3H), 3.30-2.94 (m, 4H), 2.73 (s,
3H), 2.30 (s, 3H), 2.13 (s, 3H). ESI-MS calculated for
C.sub.28H.sub.28N.sub.5O.sub.4 [M+H].sup.+=498.21; Observed:
498.33
##STR00464##
3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)amino)-3-(3-fluorophenyl)propanoic acid (Cpd. No. 239)
S13 (68 mg), 3-amino-3-(3-fluorophenyl)propanoic acid (80 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 239 as a CF.sub.3CO.sub.2H
salt in 48 mg (41% yield). .sup.1H NMR (300 MHz, MeOD-d.sub.4):
8.00 (s, 1H), 7.46 (s, 1H), 7.42-7.24 (m, 3H), 7.10-6.96 (m, 1H),
6.13 (t, J=6.00 Hz, 1H), 3.97 (s, 3H), 3.23 (d, J=6.1 Hz, 1H) 2.68
(s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS calculated for
C.sub.26H.sub.25FN.sub.5O.sub.4 [M+H].sup.+=490.19; Observed:
490.17.
##STR00465##
2-Cyclohexyl-2-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimi-
do[4,5-b]indol-4-yl)amino)acetic acid (Cpd. No. 240)
S13 (68 mg), 2-amino-2-cyclohexylacetic acid (80 mg), NaHCO.sub.3
(100 mg) and anhydrous DMSO (3 mL) were heated at 130.degree. C.
for overnight. The mixture was then purified by reverse phase HPLC
to yield Cpd. No. 240 as a CF.sub.3CO.sub.2H salt in 47 mg (41%
yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.93 (s, 1H), 7.45 (s, 1H),
5.05 (d, J=7.32 Hz, 1H), 3.97 (s, 3H), 3.30 (s, 3H), 2.72 (s, 3H),
2.33 (s, 3H), 2.30-2.10 (m, 1H), 2.16 (s, 3H), 2.04-1.90 (m, 2H),
1.90-1.64 (m, 3H), 1.50-1.10 (m, 5H). ESI-MS calculated for
C.sub.25H.sub.30N.sub.5O.sub.4 [M+H].sup.+=464.23; Observed:
464.33.
##STR00466##
cis-1-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b-
]indol-4-yl)amino)-2,3-dihydro-1H-indene-2-carboxylic acid (Cpd.
No. 241)
S13 (68 mg), cis-1-amino-2,3-dihydro-1H-indene-2-carboxylic acid
(80 mg), NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated
at 130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 241 as a CF.sub.3CO.sub.2H
salt in 47 mg (28% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.81 (s,
1H), 7.44 (s, 1H), 7.40-7.20 (m, 4H), 6.43 (d, J=7.68 Hz, 1H),
3.90-3.80 (m, 1H), 3.89 (s, 3H), 3.59 (dd, J=16.21, 4.90 Hz, 1H),
3.42-3.30 (m, 1H), 2.78 (s, 3H), 2.30 (s, 3H), 2.13 (s, 3H). ESI-MS
calculated for C.sub.27H.sub.26N.sub.5O.sub.4 [M+H].sup.+=484.20;
Observed: 484.42.
##STR00467##
(2S)-2-Cyclopentyl-2-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-
-pyrimido[4,5-b]indol-4-yl)amino)acetic acid (Cpd. No. 242)
S13 (68 mg), (S)-2-amino-2-cyclopentylacetic acid (56 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 242 as a CF.sub.3CO.sub.2H
salt in 47 mg (38% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.99 (s,
1H), 7.44 (s, 1H), 4.97 (d, J=9.56 Hz, 1H), 3.97 (s, 3H), 2.76-7.62
(m, 1H), 2.70 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H), 2.06-1.90 (m,
2H), 1.84-1.42 (m, 6H). ESI-MS calculated for
C.sub.24H.sub.28N.sub.5O.sub.4 [M+H].sup.+=450.21; Observed:
450.33.
##STR00468##
4-(4-(Isoindolin-2-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,-
5-dimethyl-isoxazole (Cpd. No. 243)
S13 (68 mg), isoindoline (70 mg), NaHCO.sub.3 (100 mg) and
anhydrous DMSO (3 mL) were heated at 130.degree. C. for overnight.
The mixture was then purified by reverse phase HPLC to yield Cpd.
No. 243 as a CF.sub.3CO.sub.2H salt in 5 mg (5% yield). .sup.1H NMR
(300 MHz, MeOD-d4): 8.04 (s, 1H), 7.60-7.50 (m, 2H), 7.46 (s, 1H),
7.45-7.35 (m, 2H), 5.68 (s, 4H), 4.03 (s, 3H), 2.76 (s, 3H), 2.36
(s, 3H), 2.20 (s, 3H). ESI-MS calculated for
C.sub.25H.sub.24N.sub.5O.sub.2 [M+H].sup.+=426.19; Observed:
426.42.
##STR00469##
2-Cyclobutyl-2-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrim-
ido[4,5-b]indol-4-yl)amino)acetic acid (Cpd. No. 244)
S13 (68 mg), 2-amino-2-cyclobutylacetic acid (60 mg), NaHCO.sub.3
(100 mg) and anhydrous DMSO (3 mL) were heated at 130.degree. C.
for overnight. The mixture was then purified by reverse phase HPLC
to yield Cpd. No. 244 as a CF.sub.3CO.sub.2H salt in 32 mg (29%
yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.00 (s, 1H), 7.44 (s, 1H),
5.13 (d, J=10.19 Hz, 1H), 3.96 (s, 3H), 3.30-3.00 (m, 1H), 2.71 (s,
3H), 2.40-2.10 (m, 3H), 2.32 (s, 3H), 2.10-1.80 (m, 3H), 2.15 (s,
3H). ESI-MS calculated for C.sub.23H.sub.26N.sub.5O.sub.4
[M+H]=436.20; Observed: 436.58.
##STR00470##
(3R)-3-(4-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)propanoic acid (Cpd. No.
245)
S13 (68 mg), (R)-3-amino-3-(4-chlorophenyl)propanoic acid (80 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 245 as a CF.sub.3CO.sub.2H
salt in 35 mg (29% yield). .sup.1H NMR (300 MHz, MeOD-d4): 83.00
(s, 1H), 7.51 (d, 1H, J=8.6 Hz), 7.46 (s, 1H), 7.36 (d, 1H, J=8.5
Hz), 6.11 (t, J=5.95 Hz, 1H), 3.97 (s, 3H), 3.22 (d, J=6.06 Hz,
2H), 2.68 (s, 3H), 2.31 (s, 3H), 2.14 (s, 3H). ESI-MS calculated
for C.sub.26H.sub.25.sup.35ClN.sub.5O.sub.4 [M+H].sup.+=506.16;
Observed: 506.67.
##STR00471##
4-(Dimethylamino)-1-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-p-
yrimido[4,5-b]indol-4-yl)pyridin-1-ium 2,2,2-trifluoroacetate (Cpd.
No. 246)
S13 (70 mg), 3-methyl-4-phenyl-1H-pyrozol-5-amine (40 mg),
EtN(i-Pr).sub.2 (0.2 mL), 4-dimethylaminopyridine (4 mg), and
anhydrous DMSO (3 mL) were heated at 130.degree. C. for overnight.
The mixture was then purified by reverse phase HPLC to yield Cpd.
No. 246 in 14 mg (78% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.86
(d, J=8.02 Hz, 2H), 7.48 (s, 1H), 7.37 (d, J=8.02 Hz, 2H), 7.34 (s,
1H), 3.83 (s, 3H), 3.45 (s, 6H), 2.83 (s, 3H), 2.32 (s, 3H), 2.14
(s, 3H). ESI-MS calculated for C.sub.24H.sub.25N.sub.6O.sub.2
[M].sup.+=429.20; Observed: 429.42.
##STR00472##
tert-Butyl
((2S)-2-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-py-
rimido[4,5-b]indol-4-yl)amino)-2-phenylethyl)carbamate (CE214)
S13 (684 mg), amine (708 mg), NaHCO.sub.3 (600 mg) and anhydrous
DMSO (10 mL) were heated at 130.degree. C. for 16 h. The mixture
was diluted with water and the aqueous layer was extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried and concentrated on a rotary evaporator. The remaining
residue was then purified by flash column chromatography to yield
CE214 in 320 mg (30% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.90
(s, 1H), 7.48-7.40 (m, 2H), 7.38-7.20 (m, 4H), 5.62-5.50 (m, 1H),
5.26-5.10 (m, 1H), 3.90-3.74 (m, 1H), 3.58-3.46 (m, 1H), 2.58 (s,
3H), 2.34 (s, 3H), 2.21 (s, 3H), 1.38 (s, 9H). ESI-MS calculated
for C.sub.30H.sub.35N.sub.6O.sub.4 [M+H].sup.+=543.27; observed:
543.33.
##STR00473##
N-((2S)-2-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,-
5-b]indol-4-yl)amino)-2-phenylethyl)-1-methylpiperidine-4-carboxamide
(Cpd. No. 247)
Step 1: CE214 (120 mg, 0.22 mmol) was dissolved in
CH.sub.2Cl.sub.2--CF.sub.3CO.sub.2H (10 mL 2:3) followed by
addition of triethylsilane (0.1 mL). The reaction mixture was
stirred at ambient temperature for 2 h. The volatile components
were removed on a rotary evaporator and the remaining residue was
used for next step without further purification.
Step 2: The previous crude residue from step
1,1-methylpiperidine-4-carboxylic acid (90 mg, 0.6 mmol), EDCI-HCl
(191 mg, 1 mmol) and HOBt (135 mg, 1 mmol) were dissolved in
anhydrous DMF (3 mL) followed by addition of EtN(i-Pr).sub.2 (0.5
mL). The reaction mixture was stirred at ambient temperature for
overnight and the mixture was then purified by reverse phase HPLC
to yield the title compound as a salt of CF.sub.3CO.sub.2H in 80 mg
(59% e yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.32 (s, 1H), 7.45
(s, 1H), 7.45-7.24 (m, 5H), 5.77 (dd, J=5.97, 3.71 Hz, 1H), 4.11
(s, 3H), 3.92-3.80 (m, 2H), 3.57-3.46 (m, 2H), 3.04-2.90 (m, 2H),
2.82 (s, 3H), 2.56-2.44 (m, 1H), 2.56 (s, 3H), 2.33 (s, 3H), 2.16
(s, 3H), 2.04-1.82 (m, 4H). ESI-MS calculated for
C.sub.32H.sub.38N.sub.7O.sub.3 [M+H].sup.+=568.30; observed:
568.33.
##STR00474##
(1S)--N1-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-
-b]indol-4-yl)-N2-(1-methylpiperidin-4-yl)-1-phenylethane-1,2-diamine
(Cpd. No. 248)
Step 1: Cpd. No. 228 (70 mg, 0.158 mmol) was dissolved in DMSO (5
mL) followed by addition of IBX (200 mg). The mixture was stirred
at ambient temperature for overnight. NaHCO.sub.3 saturate solution
was added and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried and
concentrated on a rotary evaporator. The remaining residue
containing
(2S)-2-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-2-phenylacetaldehyde was used directly for the
next step.
Step 2: the previous crude aldehyde residue from step 1,
N-methylpiperidin-4-amine (60 mg) and acetic acid (0.2 mL) were
dissolved in anhydrous THF (4 mL) followed by addition of
NaBH(OAc).sub.3 (400 mg, 2 mmol). The reaction mixture was stirred
at ambient temperature for overnight. The volatile components were
removed on a rotary evaporator and the residue was purified on
reverse phase HPLC to yield the title compound Cpd. No. 248 as a
salt of CF.sub.3CO.sub.2H in 55 mg (53% yield). .sup.1H NMR (300
MHz, MeOD-d4): 8.11 (s, 1H), 7.68-7.60 (m, 2H), 7.48-7.30 (m, 3H),
7.43 (s, 1H), 6.44 (dd, J=10.66, 3.07 Hz, 1H), 4.10 (t, J=11.86 Hz,
1H), 3.96 (s, 3H), 3.77 (dd, J=12.87, 3.43 Hz, 1H), 3.72-3.58 (m,
3H), 3.22-3.06 (m, 2H), 2.88 (s, 3H), 2.72 (s, 3H), 2.58-2.40 (m,
2H), 2.31 (s, 3H), 2.14 (s, 3H), 2.12-2.00 (m, 2H). ESI-MS
calculated for C.sub.31H.sub.38N.sub.7O.sub.2 [M+H].sup.+=540.31;
observed: 540.33.
##STR00475##
7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-N--((S)-2-(4-methylpipera-
zin-1-yl)-1-phenylethyl)-9H-pyrimido[4,5-b]indol-4-amine (Cpd. No.
249)
Following the method for the preparation of Cpd. No. 248, 80 mg of
(2S)-24(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-2-phenylacetaldehyde was prepared and placed in
a round-bottom flask. 1-Methylpiperazine (60 mg, 0.6 mmol), acetic
acid (0.1 mL), and NaBH(OAc).sub.3 (212 mg, 1.0 mmol) were
subsequently added and the reaction mixture was stirred at ambient
temperature for overnight. The volatile components were removed on
a rotary evaporator and the residue was purified on reverse phase
HPLC to yield the title compound Cpd. No. 249 as a salt of
CF.sub.3CO.sub.2H in 22 mg (17% yield). CE222 was also isolated as
a salt of CF.sub.3CO.sub.2H in 30 mg (37% yield). .sup.1H NMR (300
MHz, MeOD-d4): 8.05 (s, 1H), 7.62-7.55 (m, 2H), 7.44 (s, 1H),
7.45-7.30 (m, 3H), 6.28 (dd, J=10.60, 4.35 Hz, 1H), 3.96 (s, 3H),
3.68 (dd, J=13.14, 10.70 Hz, 1H), 3.50-3.22 (m, 9H), 2.88 (s, 3H),
2.70 (s, 3H), 2.31 (s, 3H), 2.14 (s, 3H). ESI-MS calculated for
C.sub.30H.sub.36N.sub.7O.sub.2 [M+H].sup.+=526.29; observed:
526.58.
##STR00476##
7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-4-
-amine (CE222)
.sup.1H NMR (300 MHz, MeOD-d4): 8.00 (s, 1H), 7.41 (s, 1H), 3.95
(s, 3H), 2.69 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H). ESI-MS
calculated for C.sub.17H.sub.18N.sub.5O.sub.2 [M+H].sup.+=324.15;
observed: 324.25.
##STR00477##
7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-N--((S)-2-morpholino-1-ph-
enylethyl)-9H-pyrimido[4,5-b]indol-4-amine (Cpd. No. 250)
Following the method for the preparation of Cpd. No. 248, 80 mg of
(2S)-24(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-2-phenylacetaldehyde was prepared and placed in
a round-bottom flask. Morpholine (54 mg, 0.6 mmol), acetic acid
(0.1 mL), and NaBH(OAc).sub.3 (212 mg, 0.5 mmol) were subsequently
added and the reaction mixture was stirred at ambient temperature
for overnight. The volatile components were removed on a rotary
evaporator and the residue was purified on reverse phase HPLC to
yield the title compound Cpd. No. 250 as a salt of
CF.sub.3CO.sub.2H in 19 mg (15% yield). CE222 was also isolated as
side product. .sup.1H NMR (300 MHz, MeOD-d4): 8.06 (s, 1H),
7.64-7.53 (m, 2H), 7.50-7.35 (m, 3H), 7.43 (s, 1H), 6.66 (dd,
J=11.63, 2.98 Hz, 1H), 4.17 (dd, J=13.25, 11.83 Hz, 1H), 3.98-3.83
(m, 5H), 3.96 (s, 3H), 3.80-3.55 (m, 2H), 3.55-3.40 (m, 2H), 2.73
(s, 3H), 2.31 (s, 3H), 2.14 (s, 3H). ESI-MS calculated for
C.sub.29H.sub.33N.sub.6O.sub.3 [M+H].sup.+=513.26; observed:
513.17.
##STR00478##
(3R)-3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-N-(1-methylpiperidin-4-yl)-3-phenylpropanamide
(Cpd. No. 251)
Cpd. No. 251 was prepared from Cpd. No. 232 (46 mg) and
N-methylpiperidin-4-amine (40 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 251 as a CF.sub.3CO.sub.2H
salt in 51 mg (75% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.17 (s,
1H), 7.47 (s, 1H), 7.47-7.42 (m, 2H), 7.38-7.25 (m, 3H), 5.95 (t,
J=4.72 Hz, 1H), 4.03 (s, 1H), 4.00-3.82 (m, 1H), 3.55-3.40 (m, 2H),
3.14-2.90 (m, 4H), 2.82 (s, 1H), 2.63 (s, 1H), 2.34 (s, 1H), 2.17
(s, 1H), 2.12-1.80 (m, 2H), 1.74-1.58 (m, 1H), 1.54-1.36 (m, 1H).
ESI-MS calculated for C.sub.32H.sub.38N.sub.7O.sub.3
[M+H].sup.+=568.30; observed: 568.25.
##STR00479##
(3R)-3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-1-(4-methylpiperazin-1-yl)-3-phenylpropan-1-one
(Cpd. No. 252)
Cpd. No. 252 was prepared from Cpd. No. 232 (40 mg) and
1-methylpiperazine (30 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 252 as a CF.sub.3CO.sub.2H salt in 40
mg (70% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.07 (s, 1H),
7.56-7.48 (m, 2H), 7.46 (s, 1H)), 7.46-7.26 (m, 3H), 6.09 (t,
J=4.30 Hz, 1H), 4.80-4.60 (m, 1H), 4.20-4.00 (m, 1H), 4.01 (s, 3H),
3.58 (dd, J=15.73, 4.78 Hz, 1H), 3.53-2.60 (m, 8H), 2.46 (s, 3H),
2.32 (s, 3H), 2.15 (s, 3H). ESI-MS calculated for
C.sub.31H.sub.36N.sub.7O.sub.3 [M+H].sup.+=554.29; observed:
554.33.
##STR00480##
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)-N-ethylpropanamide (Cpd. No.
253)
Cpd. No. 253 was prepared from Cpd. No. 236 (30 mg) and ethyl amine
(0.4 mmol, 0.2 mL of 2M solution in THF) using general amide
condensation method promoted by EDCI-HCl. The reaction mixture was
purified by reverse phase HPLC to yield Cpd. No. 253 as a
CF.sub.3CO.sub.2H salt in 33 mg (85% yield). .sup.1H NMR (300 MHz,
MeOD-d4): 8.19 (s, 1H), 7.49 (s, 2H), 7.45-7.20 (m, 3H), 5.92 (t,
J=4.69 Hz, 1H), 4.06 (s, 3H), 3.30-3.10 (m, 2H), 3.10 (dd, J=14.69,
5.09 Hz, 1H), 2.91 (dd, J=14.69, 4.52 Hz, 1H), 2.66 (s, 3H), 2.36
(s, 3H), 2.19 (s, 3H), 1.02 (t, J=7.26 Hz, 3H). ESI-MS calculated
for C.sub.28H.sub.30.sup.35ClN.sub.6O.sub.3 [M+H].sup.+=533.21;
Observed: 533.62.
##STR00481##
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)-1-(4-methylpiperazin-1-yl)propan-1--
one (Cpd. No. 254)
Cpd. No. 254 was prepared from Cpd. No. 236 (40 mg) and
1-methylpiperazine (30 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 254 as a CF.sub.3CO.sub.2H salt in 41
mg (72% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.02 (s, 1H),
7.60-7.56 (m, 1H), 7.50-7.28 (m, 3H), 7.46 (s, 1H), 6.11 (t, J=4.99
Hz, 1H), 4.00 (s, 3H), 3.70-2.80 (br, 8H), 3.56 (dd, J=16.14, 5.42
Hz, 1H), 3.24 (dd, J=16.14, 4.97 Hz, 1H), 2.67 (s, 3H), 2.32 (s,
3H), 2.15 (s, 3H). ESI-MS calculated for
C.sub.31H.sub.35.sup.35ClN.sub.7O.sub.3 [M+H].sup.+=588.25;
Observed: 588.58.
##STR00482##
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)-1-morpholinopropan-1-one
(Cpd. No. 255)
Cpd. No. 255 was prepared from Cpd. No. 236 (40 mg) and morpholine
(27 mg) using general amide condensation method promoted by
EDCI-HCl. The reaction mixture was purified by reverse phase HPLC
to yield Cpd. No. 255 as a CF.sub.3CO.sub.2H salt in 31 mg (56%
yield). .sup.1H NMR (300 MHz, MeOD-d.sub.4): 8.07 (s, 1H),
7.56-7.26 (m, 4H), 7.47 (s, 1H), 6.02 (t, J=4.73 Hz, 1H), 4.01 (s,
3H), 3.70-3.20 (m, 8H), 3.20-3.00 (m, 2H), 2.65 (s, 3H), 2.32 (s,
3H), 2.15 (s, 3H). ESI-MS calculated for
C.sub.30H.sub.32.sup.35ClN.sub.6O.sub.4 [M+H].sup.+=575.22;
Observed: 575.62.
##STR00483##
2-Cyclohexyl-2-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrim-
ido[4,5-b]indol-4-yl)amino)-1-(4-methylpiperazin-1-yl)ethanone
(Cpd. No. 256)
Cpd. No. 256 was prepared from Cpd. No. 240 (36 mg) and
1-methylpiperazine (38 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 256 as a CF.sub.3CO.sub.2H salt in 17
mg (33% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.99 (s, 1H), 7.48
(s, 1H), 4.00 (s, 3H), 5.70-5.50 (m, 1H), 3.90-3.00 (m, 8H), 2.98
(s, 3H), 2.77 (s, 3H), 2.34 (s, 3H), 2.30-2.10 (m, 1H), 2.17 (s,
3H), 2.10-1.95 (m, 1H), 1.95-1.65 (m, 4H), 1.50-1.10 (m, 5H).
ESI-MS calculated for C.sub.30H.sub.40N.sub.7O.sub.3
[M+H].sup.+=546.32; Observed: 546.42.
##STR00484##
(2S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-N-(1-methylpiperidin-4-yl)-2-phenylacetamide
(Cpd. No. 257)
Step 1: Fmoc-L-phenylglycine (740 mg, 2.0 mmol), EDCI-HCl (600 mg,
3.0 mmol) and HOBt (405 mg, 3.0 mmol) were dissolved in
dichloromethane (10 mL). EtN(i-Pr).sub.2 (0.5 mL) and
N-methylpiperidin-4-amine (228 mg, 2.0 mmol) were sequentially
added via syringes. The mixture was stirred at ambient temperature
for 4 h. Water was added and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined, dried and
concentrated on a rotary evaporator. The remaining residue was used
directly for the next step.
Step 2: The previous residue from step 1 was dissolved in THF (10
mL). EtNH.sub.2 (5 mL, 2.0 M in THF) was added via a syringe and
the mixture was stirred for 12 h. The volatile components were
removed on a rotary evaporator. The remaining residue was vacuumed
for 1 day and used directly for the next step.
Step 3: The previous residue from step 2 was dissolved in anhydrous
DMSO (6 mL). NaHCO.sub.3 (200 mg) and S13 (400 mg, 1.17 mmol) were
added and the mixture was heated at 130.degree. C. for overnight.
Water (2 mL) was added and the mixture was filtered. The solution
was purified by reverse phase HPLC to yield the title compound Cpd.
No. 257 as a salt of CF.sub.3CO.sub.2H in 55 mg (7% yield). .sup.1H
NMR (300 MHz, MeOD-d4): 7.87 (s, 1H), 7.68-7.60 (m, 2H), 7.54 (s,
1H), 7.54-7.34 (m, 3H), 6.13 (s, 1H), 4.12-4.00 (m, 1H), 3.95 (s,
3H), 3.64-3.48 (m, 2H), 3.26-3.00 (m, 3H), 2.86 (s, 3H), 2.70 (s,
3H), 2.31 (s, 3H), 2.30-2.00 (m, 2H), 2.14 (s, 3H), 1.96-1.66 (m,
2H). ESI-MS calculated for C.sub.31H.sub.36N.sub.7O.sub.3
[M+H].sup.+=554.29; observed: 554.17.
##STR00485##
(2S,3S)-2-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4-
,5-b]indol-4-yl)amino)-3-methyl-N-(1-methylpiperidin-4-yl)pentanamide
(Cpd. No. 258)
Cpd. No. 258 was prepared from Cpd. No. 231 (43 mg) and
N-methylpiperidin-4-amine (36 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 258 as a CF.sub.3CO.sub.2H
salt in 15 mg (23% yield). ESI-MS calculated for
C.sub.29H.sub.40N.sub.7O.sub.3 [M+H].sup.+=534.32; observed:
534.08.
4-(6-Methoxy-2-methyl-4-(4-((4-(methylsulfonyl)piperazin-1-yl)methyl)napht-
halen-1-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole
(Cpd. No. 259)
##STR00486##
CE194 (46 mg), 1-(methylsulfonyl)piperazine (48 mg, 0.3 mmol), and
acetic acid (0.1 mL) were dissolved in anhydrous THF (5 mL).
NaBH(OAc).sub.3 (110 mg, 0.5 mmol) was added in one portion and the
mixture was stirred at ambient temperature for overnight. The
volatile components were removed on a rotary evaporator. The
remaining residue was purified by reverse phase HPLC to yield the
title compound Cpd. No. 259 as a CF.sub.3CO.sub.2H salt in 71 mg
(98% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.31 (d, J=8.47 Hz,
1H), 8.16 (d, J=7.40 Hz, 1H), 8.09 (d, J=7.38 Hz, 1H), 7.90-7.82
(m, 2H), 7.72-7.63 (m, 1H), 7.55 (s, 1H), 6.20 (s, 1H), 5.15 (d,
J=13.60 Hz, 1H), 5.09 (d, J=13.74 Hz, 1H), 3.18 (s, 3H), 3.02 (s,
3H), 2.94 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated
for C.sub.33H.sub.35N.sub.6O.sub.4S [M+H].sup.+=611.24, Observed:
611.58.
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-N-methyl-1H-pyrrole-2-carboxamide (Cpd. No.
260)
##STR00487##
Cpd. No. 260 was prepared from Cpd. No. 210 (46 mg) and methyl
amine-HCl (21 mg) using general amide condensation method promoted
by EDCI-HCl. The reaction mixture was purified by reverse phase
HPLC to yield Cpd. No. 260 as a CF.sub.3CO.sub.2H salt in 38 mg
(65% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.54 (s, 1H), 7.29 (s,
1H), 7.16 (s, 1H), 3.72 (s, 3H), 3.14 (septet, J=6.99 Hz, 1H), 2.93
(s, 3H), 2.92 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H), 1.28 (d, J=6.97
Hz, 6H). ESI-MS calculated for C.sub.26H.sub.29N.sub.6O.sub.3
[M+H].sup.+=473.23, Observed: 473.44.
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)-5-isopropyl-1H-pyrrol-2-yl)(4-hydroxypiperidin-1-yl)methanone
(Cpd. No. 261)
##STR00488##
Cpd. No. 261 was prepared from Cpd. No. 210 (35 mg) and
4-hydroxylpiperidine (30 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 261 as a CF.sub.3CO.sub.2H
salt in 22 mg (45% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.54 (s,
1H), 7.25 (s, 1H), 6.93 (s, 1H), 4.30-4.18 (m, 2H), 4.00-3.88 (m,
1H), 3.73 (s, 3H), 3.60-3.40 (m, 2H), 3.11 (septet, J=6.98 Hz, 1H),
2.93 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H), 2.00-1.88 (m, 2H),
1.62-1.46 (m, 2H), 1.28 (d, J=6.98 Hz, 6H). ESI-MS calculated for
C.sub.30H.sub.35N.sub.6O.sub.4 [M+H].sup.+=543.27, Observed:
543.92.
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(4-hydroxycyclohexyl)-5-isopropyl-1H-pyrrole-2-carboxamide
(Cpd. No. 262)
##STR00489##
Cpd. No. 262 was prepared from Cpd. No. 210 (35 mg) and
trans-4-aminohexanol (30 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 262 as a CF.sub.3CO.sub.2H
salt in 28 mg (55% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.54 (s,
1H), 7.31 (s, 1H), 7.26 (s, 1H), 3.94-3.80 (m, 1H), 3.72 (s, 3H),
3.60-3.48 (m, 1H), 3.16 (septet, J=6.97 Hz, 1H), 2.93 (s, 3H), 2.31
(s, 3H), 2.13 (s, 3H), 2.08-1.92 (m, 4H), 1.54-1.36 (m, 4H), 1.29
(d, J=6.96 Hz, 6H). ESI-MS calculated for
C.sub.31H.sub.37N.sub.6O.sub.4 [M+H].sup.+=557.29, Observed:
557.33.
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-5-isopropyl-N-(oxetan-3-yl)-1H-pyrrole-2-carboxamide (Cpd.
No. 263)
##STR00490##
Cpd. No. 263 was prepared from Cpd. No. 210 (35 mg) and
oxetan-3-amine (30 mg) using general amide condensation method
promoted by EDCI-HCl. The reaction mixture was purified by reverse
phase HPLC to yield Cpd. No. 263 as a CF.sub.3CO.sub.2H salt in 18
mg (38% yield). .sup.1H NMR (300 MHz, MeOD-d4): 7.80 (s, 1H), 7.56
(s, 1H), 7.11 (s, 1H), 5.15 (t, J=9.55 Hz, 1H), 5.01 (dd, J=9.18,
6.10 Hz, 1H), 4.75-4.65 (m, 1H), 3.87 (dd, J=11.97, 2.81 Hz, 1H),
3.76 (dd, J=11.97, 3.40 Hz, 1H), 3.71 (s, 3H), 3.20 (septet, J=7.01
Hz, 1H), 2.94 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H), 1.32 (d, J=6.98
Hz, 6H). ESI-MS calculated for C.sub.28H.sub.31N.sub.6O.sub.4
[M+H].sup.+=515.24, Observed: 515.25.
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-((cis)-3-hydroxy-3-methylcyclobutyl)-5-isopropyl-1H-pyrrole-2-ca-
rboxamide (Cpd. No. 264)
##STR00491##
Cpd. No. 210 (35 mg) and
(cis)-3-((tert-butyldimethylsilyl)oxy)-3-methylcyclobutanamine (35
mg) were coupled using general amide condensation method promoted
by EDCI-HCl. The reaction mixture was treated with
CF.sub.3CO.sub.2H (4 mL) and was stirred at room temperature for 2
hours. The mixture was purified by reverse phase HPLC to yield Cpd.
No. 264 as a CF.sub.3CO.sub.2H salt in 42 mg (72% yield). .sup.1H
NMR (300 MHz, MeOD-d4): 7.54 (s, 1H), 7.30 (s, 1H), 7.27 (s, 1H),
4.11 (quintet, J=8.30 Hz, 1H), 3.72 (s, 3H), 3.15 (septet, J=6.97
Hz, 1H), 2.93 (s, 3H), 2.56-2.42 (m, 2H), 2.31 (s, 3H), 2.20-2.08
(m, 2H), 2.13 (s, 3H), 1.39 (s, 3H), 1.29 (d, J=6.97 Hz, 6H).
ESI-MS calculated for C.sub.30H.sub.35N.sub.6O.sub.4
[M+H].sup.+=543.27, Observed: 543.50.
(4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]ind-
ol-4-yl)naphthalen-1-yl)(4-hydroxypiperidin-1-yl)methanone (Cpd.
No. 265)
##STR00492##
Cpd. No. 265 was prepared from the acid Cpd. No. 165 (48 mg) and
4-hydroxylpiperidine (30 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 265 as a CF.sub.3CO.sub.2H
salt in 60 mg (89% yield). .sup.1H NMR (300 MHz, MeOD-d4):
8.16-8.04 (m, 2H), 7.90-7.74 (m, 3H), 7.70-7.60 (m, 1H), 7.58-7.52
(m, 1H), 6.28-6.10 (m, 1H), 4.50-4.30 (m, 1H), 4.10-3.90 (m, 1H),
3.70-3.50 (m, 1H), 3.50-3.35 (m, 1H), 3.21 (s, 3H), 3.03 (s, 3H),
3.02 (s, 3H), 2.26 (s, 3H), 2.20-2.00 (m, 1H), 2.06 (s, 3H),
1.90-1.60 (m, 2H), 1.60-1.20 (m, 1H). ESI-MS calculated for
C.sub.33H.sub.32N.sub.5O.sub.4 [M+H].sup.+=562.25, Observed:
562.67.
4-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indo-
l-4-yl)-N-(4-hydroxycyclohexyl)-1-naphthamide (Cpd. No. 266)
##STR00493##
Cpd. No. 266 was prepared from the acid Cpd. No. 165 (48 mg) and
trans-4-aminohexanol (40 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 266 as a CF.sub.3CO.sub.2H
salt in 37 mg (53% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.41 (d,
J=8.44 Hz, 1H), 8.02 (d, J=7.33 Hz, 1H), 7.89 (d, J=7.28 Hz, 1H),
7.83-7.73 (m, 2H), 7.65-7.58 (m, 1H), 7.53 (s, 1H), 6.17 (s, 1H),
4.10-4.00 (m, 1H), 3.70-3.55 (m, 1H), 3.19 (s, 3H), 3.01 (s, 3H),
2.24-2.12 (m, 2H), 2.10-2.02 (m, 2H), 2.26 (s, 3H), 2.07 (s, 3H),
1.56-1.44 (m, 4H). ESI-MS calculated for
C.sub.34H.sub.34N.sub.5O.sub.4 [M+H].sup.+=576.26, Observed:
576.58.
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)-1-(4-hydroxypiperidin-1-yl)propan-1-
-one (Cpd. No. 267)
##STR00494##
Cpd. No. 267 was prepared from the acid Cpd. No. 236 (40 mg) and
4-hydroxylpiperidine (30 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 267 as a CF.sub.3CO.sub.2H
salt in 34 mg (60% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.08 (s,
1H), 7.60-7.20 (m, 4H), 7.47 (s, 1H), 6.10-5.90 (m, 1H), 4.20-3.65
(m, 3H), 4.02 (s, 3H), 3.60-3.40 (m, 1H), 3.40-3.00 (m, 3H), 2.66
(s, 3H), 2.33 (s, 3H), 2.17 (s, 3H), 1.90-1.60 (m, 1.5H), 1.50-1.30
(m, 1.5H), 1.30-1.10 (m, 0.5H), 1.00-0.80 (m, 0.5H). ESI-MS
calculated for C.sub.31H.sub.34.sup.35ClN.sub.6O.sub.4
[M+H].sup.+=589.23, Observed: 589.58.
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)-N-((trans)-4-hydroxycyclohexyl)prop-
anamide (Cpd. No. 268)
##STR00495##
Cpd. No. 268 was prepared from the acid Cpd. No. 236 (40 mg) and
4-hydroxylpiperidine (33 mg) using general amide condensation
method promoted by EDCI-HCl. The reaction mixture was purified by
reverse phase HPLC to yield Cpd. No. 268 as a CF.sub.3CO.sub.2H
salt in 52 mg (90% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.18 (s,
1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.42-7.30 (m, 3H), 5.89 (t, J=4.70
Hz, 1H), 4.04 (s, 3H), 3.70-3.54 (m, 1H), 3.54-3.40 (m, 1H), 3.05
(dd, J=14.45, 5.15 Hz, 1H), 2.87 (dd, J=14.51, 4.50 Hz, 1H), 2.63
(s, 3H), 2.33 (s, 3H), 2.17 (s, 3H), 2.00-1.60 (m, 4H), 1.40-0.90
(m, 4H). ESI-MS calculated for
C.sub.32H.sub.36.sup.35ClN.sub.6O.sub.4 [M+H].sup.+=603.25,
Observed: 603.58.
(3R)-3-((7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5--
b]indol-4-yl)amino)-3-(3-methoxyphenyl)propanoic acid (Cpd. No.
269)
##STR00496##
S13 (70 mg), (R)-3-amino-3-(3-methoxyphenyl)propanoic acid (80 mg),
NaHCO.sub.3 (100 mg) and anhydrous DMSO (3 mL) were heated at
130.degree. C. for overnight. The mixture was then purified by
reverse phase HPLC to yield Cpd. No. 269 as a CF.sub.3CO.sub.2H
salt in 39 mg (32% yield). .sup.1H NMR (300 MHz, MeOD-d4): 8.00 (s,
1H), 7.46 (s, 1H), 7.32-7.22 (m, 1H), 7.12-7.04 (m, 2H), 6.88-6.82
(m, 1H), 6.08 (t, J=5.89 Hz, 1H), 3.97 (s, 3H), 3.77 (s, 3H), 3.21
(d, J=5.95 Hz, 2H), 2.68 (s, 3H), 2.13 (s, 3H), 2.15 (s, 3H).
ESI-MS calculated for C.sub.27H.sub.28N.sub.5O.sub.5
[M+H].sup.+=502.21, Observed: 502.34.
(3R)-3-(3-Chlorophenyl)-3-((7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-meth-
yl-9H-pyrimido[4,5-b]indol-4-yl)amino)propan-1-ol (Cpd. No.
270)
Step 1: The acid Cpd. No. 236 (220 mg) was dissolved in MeOH (20
mL). Four drops of concentrated H.sub.2SO.sub.4 was added via a
glass pipet. The reaction mixture was heated at reflux for
overnight. The reaction solution was concentrated and treated with
NaHCO.sub.3 saturated solution and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were combined and
dried over anhydrous sodium sulfate. The volatile components were
removed on a rotary evaporator. The remaining residue was used for
the next step without purification.
Step 2: The previous residue was dissolved in ethanol. Sodium
borohydride (200 mg) was added at ambient temperature. The mixture
was stirred at room temperature for overnight. NaHCO.sub.3
saturated aqueous solution was added and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
combined and dried over anhydrous sodium sulfate. The volatile
components were removed on a rotary evaporator. The remaining
residue was purified on a reverse phase HPLC to yield Cpd. No. 270
in 52 mg (56% yield) as a CF.sub.3CO.sub.2H salt.
##STR00497##
.sup.1H NMR (300 MHz, MeOD-d4): 7.90 (s, 1H), 7.52 (s, 1H), 7.49
(s, 1H), 7.50-7.20 (m, 3H), 5.87 (t, J=5.07 Hz, 1H), 3.97 (s, 3H),
3.94-3.84 (m, 1H), 3.84-3.72 (m, 1H), 2.64 (s, 3H), 2.60-2.42 (m,
1H), 2.34 (s, 3H), 2.30-2.14 (m, 1H), 2.17 (s, 3H). ESI-MS
calculated for C.sub.26H.sub.27.sup.35ClN.sub.5O.sub.3
[M+H].sup.+=492.18, Observed: 492.62
4-(4-(1-Isopropyl-1H-pyrazol-5-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]in-
dol-7-yl)-3,5-dimethylisoxazole (Cpd. No. 271)
##STR00498##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 1.09 g, 3.2 mmol),
(1-isopropyl-1H-pyrazol-5-yl)boronic acid (1.0 g, 6.5 mmol),
1,2-dimethoxyethane (18 mL), and Na.sub.2CO.sub.3 (2 M in water, 6
mL) were added. The system was degassed to remove oxygen and
nitrogen was refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (245 mg,
0.3 mmol) was added and the system was degassed and refilled with
nitrogen. The reaction mixture was heated at reflux for overnight.
The aqueous layer was extracted with ethyl acetate and the organic
layers were combined and the volatile components were removed on a
rotary evaporator. The residue was purified by flash column
chromatography to yield the title compound in 277 mg (21% yield).
The compound was further purified by reverse HPLC to yield Cpd. No.
271 as a CF.sub.3CO.sub.2H salt. .sup.1H NMR (300 MHz, MeOD-d4):
7.92 (d, J=1.52 Hz, 1H), 7.54 (s, 1H), 6.94 (d, J=1.81 Hz, 1H),
6.91 (s, 1H), 4.52 (septet, J=6.50 Hz, 1H), 3.69 (s, 3H), 2.94 (s,
3H), 2.31 (s, 3H), 2.13 (s, 3H), 1.46 (d, J=6.52 Hz, 6H). ESI-MS
calculated for C.sub.23H.sub.25N.sub.6O.sub.2 [M+H].sup.+=417.20,
Observed: 417.50.
2-(5-Bromo-2-methoxyphenyl)propan-2-ol (CE305)
##STR00499##
Methyl 5-bromo-2-methoxybenzoate (10 g, 40 mmol) was dissolved in
anhydrous THF (60 mL), which was subsequently cooled with ice-water
bath. MeMgBr (3.0 M in ether, 30 mL, 90 mmol) was added via a
syringe at 0.degree. C. and the reaction mixture was stirred for
overnight. The reaction was quenched with saturated ammonium
chloride. The aqueous layer was extracted with ethyl acetate and
the organic layers were combined and the volatile components were
removed on a rotary evaporator. The residue containing CE305 was
used for the next step without further purification. .sup.1H NMR
(300 MHz, CDCl.sub.3): 7.77-7.67 (m, 2H), 7.45 (d, J=2.50 Hz, 1H),
7.28 (dd, J=8.70, 2.50 Hz, 1H), 6.74 (d, J=8.71 Hz, 1H), 3.90 (s,
1H), 3.82 (s, 3H), 1.55 (s, 6H).
2-(2-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan--
2-ol (CE308)
##STR00500##
2-(5-Bromo-2-methoxyphenyl)propan-2-ol (1.0 g, 4.0 mmol),
bis(pinacolato)diboron (2.03 g, 8.0 mmol) and potassium acetate
(1.2 g, 12 mmol) were added to a round-bottom flask Anhydrous
1,4-dixoane (20 mL) was added to the flask, which was degassed and
refilled with nitrogen. Pd(dppf)Cl.sub.2 (280 mg, 0.4 mmol) was
added and the flask was degassed again followed by heating at
100.degree. C. for overnight. The reaction mixture was cooled to
room temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound was isolated in 1.27 g
contained with some impurity, which was used for the next step
without further purification. .sup.1H NMR (300 MHz, CDCl.sub.3):
6.92 (d, J=8.09 Hz, 1H), 3.93 (s, 3H), 1.63 (s, 6H), 1.33 (s, 12H).
.sup.13C NMR (75 MHz, CDCl.sub.3): 159.72, 135.75, 135.27, 132.41,
110.85, 83.84, 72.89, 55.52, 29.89, 25.06. ESI-MS calculated for
C.sub.16H.sub.25BNaO.sub.4 [M+H].sup.+=315.17, Observed:
315.67.
2-(5-(7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]i-
ndol-4-yl)-2-methoxyphenyl)propan-2-ol (Cpd. No. 272)
##STR00501##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 680 mg, 2.0 mmol), CE308 (1.27 g, 4.3 mmol),
1,2-dimethoxyethane (18 mL), and Na.sub.2CO.sub.3 (2 M in water, 6
mL) were added. The system was degassed to remove oxygen and
nitrogen was refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (163 mg,
0.2 mmol) was added and the system was degassed and refilled with
nitrogen. The reaction mixture was heated at reflux for overnight.
The aqueous layer was extracted with ethyl acetate and the organic
layers were combined and the volatile components were removed on a
rotary evaporator. The residue was purified by flash column
chromatography to yield the title compound, which was further
purified by reversed HPLC to yield Cpd. No. 272 in 221 mg (20%).
.sup.1H NMR (300 MHz, MeOD-d4): 8.14 (s, 1H), 8.82 (d, J=8.33 Hz,
1H), 7.21 (d, J=8.26 Hz, 1H), 7.44 (s, 1H), 7.32 (s, 1H), 3.99 (s,
3H), 3.71 (s, 3H), 2.78 (s, 3H), 2.31 (s, 3H), 2.14 (s, 3H), 1.66
(s, 6H). ESI-MS calculated for C.sub.27H.sub.29N.sub.4O.sub.4
[M+H].sup.+=473.22, Observed: 473.50.
##STR00502##
To a round-bottom flask, ZBA154 (0.19 g) was dissolved in THF (9
mL), t-BuOH (9 mL) and H.sub.2O (3 mL) at room temperature.
NaClO.sub.2 (303 mg), NaH.sub.2PO.sub.4 (505 mg) and
2-Methyl-2-butene (1.5 mL) was added and the reaction mixture was
stirred overnight. Then water and ethyl acetate was slowly added.
The aqueous layer was extracted with EtOAc. The combined EtOAc
extracts were washed with H.sub.2O, dried over Na.sub.2SO.sub.4,
and concentrated under reduced pressure to afford Cpd. No. 273 (140
mg) after HPLC purification. ESI-MS calculated for
C.sub.26H.sub.20N.sub.5O.sub.4 [M+H].sup.+=466.15, Obtained:
466.65.
##STR00503##
(amide condensation): ZBA191 (20 mg), HBTU (24 mg), HOBt-H.sub.2O
(6 mg) and DMF (1 mL) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.05 mL) was added followed by addition of
1-methylpiperazine (10 mg) was added and the reaction mixture was
stirred for 12 h. The reaction was quenched with NaHCO.sub.3
saturated solution and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were concentrated on a rotary
evaporator. The remaining residue was purified by reverse phase
HPLC affording the Cpd. No. 274 as a salt of CF.sub.3CO.sub.2H (14
mg). ESI-MS calculated for C.sub.31H.sub.30N.sub.7O.sub.3
[M+H].sup.+=548.24, Obtained: 548.55. .sup.1H NMR (300 MHz, MeOD)
.delta. 9.37 (d, J=5.0 Hz, 1H), 8.40 (d, J=8.5 Hz, 1H), 8.23 (d,
J=4.7 Hz, 1H), 8.12 (t, J=7.8 Hz, 1H), 8.03 (d, J=8.5 Hz, 1H),
7.84-7.72 (m, 1H), 7.50 (d, J=11.4 Hz, 1H), 6.40 (s, 1H), 4.47-4.20
(m, 1H), 3.86-3.30 (m, 8H), 3.28 (s, 3H), 2.99 (s, 3H), 2.29 (s,
3H), 2.08 (d, J=8.8 Hz, 3H).
##STR00504##
Cpd. No. 275-TFA salt was prepared from amide condensation of
ZBA191 and N,N-dimethylethylenediamine using HBTU-HOBT condition.
60% yield. ESI-MS calculated for
C.sub.30H.sub.30N.sub.7O.sub.3[M+H].sup.+=536.24, Obtained: 536.77.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.23 (d, J=4.7 Hz, 1H), 8.34
(d, J=8.5 Hz, 1H), 8.09-7.90 (m, 2H), 7.78 (d, J=8.0 Hz, 1H),
7.70-7.57 (m, 1H), 7.51 (s, 1H), 6.30 (s, 1H), 3.92 (t, J=5.6 Hz,
2H), 3.48 (d, J=5.2 Hz, 2H), 3.24 (s, 3H), 3.04 (s, 6H), 2.29 (s,
3H), 2.10 (s, 3H).
##STR00505##
Cpd. No. 276-TFA salt was prepared from amide condensation of
ZBA191 and methylamine using HBTU-HOBT condition. 60% yield. ESI-MS
calculated for C.sub.27H.sub.23N.sub.6O.sub.3[M+H].sup.+=479.18,
Obtained: 479.22. .sup.1H NMR (300 MHz, MeOD) .delta. 9.34 (d,
J=4.9 Hz, 1H), 8.38 (d, J=8.7 Hz, 1H), 8.17 (d, J=4.6 Hz, 1H), 8.08
(d, J=7.5 Hz, 1H), 7.93 (d, J=8.6 Hz, 1H), 7.75 (d, J=7.5 Hz, 1H),
7.54 (s, 1H), 6.35 (s, 1H), 3.26 (s, 3H), 3.07 (s, 3H), 2.29 (s,
3H), 2.10 (s, 3H).
##STR00506##
Cpd. No. 277-TFA salt was prepared from amide condensation of
ZBA191 and N,N-diethylethylenediamine using HBTU-HOBT condition.
70% yield. ESI-MS calculated for
C.sub.32H.sub.34N.sub.7O.sub.3[M+H].sup.+=564.27, Obtained: 564.38.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.36 (d, J=4.8 Hz, 1H), 8.40
(d, J=8.5 Hz, 1H), 8.17 (d, J=4.8 Hz, 1H), 8.08 (t, J=7.7 Hz, 1H),
7.91 (d, J=8.4 Hz, 1H), 7.74 (t, J=7.2 Hz, 1H), 7.55 (s, 1H), 6.36
(s, 1H), 3.96-3.37 (m, 8H), 3.26 (s, 3H), 2.29 (s, 3H), 2.10 (s,
3H), 1.38 (t, J=7.0 Hz, 6H).
##STR00507##
Cpd. No. 278-TFA salt was prepared from amide condensation of
ZBA191 and 1-(2-aminoethyl)pyrrolidine using HBTU-HOBT condition.
70% yield. ESI-MS calculated for
C.sub.32H.sub.32N.sub.7O.sub.3[M+H].sup.+=562.25, Obtained: 562.48.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.25 (d, J=4.6 Hz, 1H), 8.35
(d, J=8.6 Hz, 1H), 8.04-7.94 (m, 2H), 7.80 (d, J=8.2 Hz, 1H), 7.64
(t, J=7.4 Hz, 1H), 7.51 (s, 1H), 6.30 (s, 1H), 3.96-3.79 (m, 4H),
3.51 (t, J=5.4 Hz, 2H), 3.26-3.12 (m, 5H), 2.29 (s, 3H), 2.24-1.95
(m, 7H).
##STR00508##
Cpd. No. 279-TFA salt was prepared from amide condensation of
ZBA191 and 4-(2-aminoethyl)morpholine using HBTU-HOBT condition.
70% yield. ESI-MS calculated for
C.sub.32H.sub.32N.sub.7O.sub.4[M+H].sup.+=578.25, Obtained: 578.50.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.30 (d, J=4.8 Hz, 1H), 8.36
(d, J=8.4 Hz, 1H), 8.10-7.98 (m, 2H), 7.84 (d, J=7.8 Hz, 1H),
7.74-7.65 (m, 1H), 7.53 (s, 1H), 6.32 (s, 1H), 4.19-3.67 (m, 8H),
3.51 (t, J=5.7 Hz, 2H), 3.30-3.16 (m, 5H), 2.29 (s, 3H), 2.10 (s,
3H).
##STR00509##
Cpd. No. 280-TFA salt was prepared from amide condensation of
ZBA191 and 1-methyl-4-piperidinamine using HBTU-HOBT condition. 65%
yield. ESI-MS calculated for
C.sub.32H.sub.32N.sub.7O.sub.3[M+H].sup.+=562.25, Obtained: 562.66.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.38 (d, J=5.0 Hz, 1H), 8.40
(d, J=8.5 Hz, 1H), 8.20 (d, J=5.0 Hz, 1H), 8.09 (ddd, J=8.4, 7.0,
1.2 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.75 (ddd, J=8.3, 6.9, 1.0 Hz,
1H), 7.57 (s, 1H), 6.35 (s, 1H), 4.32 (tt, J=11.4, 4.0 Hz, 1H),
3.64 (d, J=13.3 Hz, 2H), 3.30-3.16 (m, 5H), 2.93 (s, 3H), 2.37-2.25
(m, 5H), 2.17-1.98 (m, 5H).
##STR00510##
ZBA191 (20 mg), HBTU (24 mg), HOBt-H.sub.2O (6 mg) and DMF (1 mL)
were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.05 mL) was
added followed by addition of 4-Amino-1-Boc-piperidine (20 mg) was
added and the reaction mixture was stirred for 12 h. The reaction
was quenched with NaHCO.sub.3 saturated solution and the aqueous
layer was extracted with ethyl acetate. The combined organic layers
were concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
281 as a salt of CF.sub.3CO.sub.2H (13 mg). ESI-MS calculated for
C.sub.31H.sub.30N.sub.7O.sub.3[M+H].sup.+=548.24, Obtained: 548.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.32 (d, J=4.8 Hz, 1H), 8.38
(d, J=8.5 Hz, 1H), 8.11 (d, J=4.8 Hz, 1H), 8.08-7.99 (m, 1H), 7.87
(d, J=8.0 Hz, 1H), 7.69 (dd, J=11.3, 4.1 Hz, 1H), 7.57 (s, 1H),
6.31 (s, 1H), 4.41-4.25 (m, 1H), 3.59-3.44 (m, 2H), 3.28-3.13 (m,
5H), 2.35-2.21 (m, 5H), 2.14-1.90 (m, 5H).
##STR00511##
ZBA191 (20 mg), HBTU (24 mg), HOBt-H.sub.2O (6 mg) and DMF (1 mL)
were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.05 mL) was
added followed by addition of (5)-1-N-Boc-2-methylpiperazine (20
mg) was added and the reaction mixture was stirred for 12 h. The
reaction was quenched with NaHCO.sub.3 saturated solution and the
aqueous layer was extracted with ethyl acetate. The combined
organic layers were concentrated on a rotary evaporator. The
remaining residue was dissolved in TFA (2 mL) and DCM (2 mL). The
mixture was stirred for 3 hours and was concentrated on a rotary
evaporator. The remaining residue was purified by reverse phase
HPLC affording the Cpd. No. 282 as a salt of CF.sub.3CO.sub.2H (13
mg). ESI-MS calculated for
C.sub.31H.sub.30N.sub.7O.sub.3[M+H].sup.+=548.24, Obtained:
548.47.
##STR00512##
ZBA191 (20 mg), HBTU (24 mg), HOBt-H.sub.2O (6 mg) and DMF (1 mL)
were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.05 mL) was
added followed by addition of t-Boc-piperazine (18 mg) was added
and the reaction mixture was stirred for 12 h. The reaction was
quenched with NaHCO.sub.3 saturated solution and the aqueous layer
was extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
283 as a salt of CF.sub.3CO.sub.2H (12 mg). ESI-MS calculated for
C.sub.30H.sub.28N.sub.7O.sub.3[M+H].sup.+=534.22, Obtained: 534.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.27 (d, J=4.7 Hz, 1H), 8.35
(d, J=8.5 Hz, 1H), 8.04-7.98 (m, 2H), 7.91 (d, J=7.7 Hz, 1H), 7.69
(ddd, J=8.3, 6.9, 1.1 Hz, 1H), 7.50 (s, 1H), 6.36 (s, 1H),
4.16-4.09 (m, 2H), 3.98-3.89 (m, 2H), 3.47-3.40 (m, 2H), 3.39-3.34
(m, 2H), 3.26 (s, 3H), 2.29 (s, 3H), 2.10 (s, 3H).
##STR00513##
Cpd. No. 284-TFA salt was prepared from amide condensation of
ZBA191 and morpholine using HBTU-HOBT condition. 75% yield. ESI-MS
calculated for C.sub.30H.sub.27N.sub.6O.sub.4[M+H].sup.+=535.20,
Obtained: 535.44. .sup.1H NMR (300 MHz, MeOD) .delta. 9.35 (d,
J=5.0 Hz, 1H), 8.39 (d, J=8.5 Hz, 1H), 8.19 (d, J=5.0 Hz, 1H), 8.10
(ddd, J=8.5, 6.9, 1.3 Hz, 1H), 8.02 (d, J=7.9 Hz, 1H), 7.78 (ddd,
J=8.3, 6.9, 1.0 Hz, 1H), 7.50 (s, 1H), 6.40 (s, 1H), 3.86 (dd,
J=7.4, 2.6 Hz, 4H), 3.78-3.69 (m, 2H), 3.66-3.57 (m, 2H), 3.29 (s,
3H), 2.29 (s, 3H), 2.10 (s, 3H).
##STR00514##
Cpd. No. 285-TFA salt was prepared from amide condensation of
ZBA191 and 4-Hydroxypiperidine using HBTU-HOBT condition. 75%
yield. ESI-MS calculated for
C.sub.31H.sub.29N.sub.6O.sub.4[M+H].sup.+=549.22, Obtained: 549.64.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.39 (s, 1H), 8.41 (d, J=8.5
Hz, 1H), 8.28 (d, J=5.0 Hz, 1H), 8.15 (t, J=7.7 Hz, 1H), 8.09 (d,
J=8.4 Hz, 1H), 7.82 (t, J=7.5 Hz, 1H), 7.50 (s, 1H), 6.45 (s, 1H),
4.35-4.15 (m, 1H), 4.03-3.68 (m, 2H), 3.55-3.35 (m, 2H), 3.31 (s,
3H), 2.29 (s, 3H), 2.10 (s, 3H), 2.07-1.87 (m, 2H), 1.72-1.55 (m,
2H).
##STR00515##
Cpd. No. 286-TFA salt was prepared from amide condensation of
ZBA191 and cis-4-Amino-cyclohexanol using HBTU-HOBT condition. 55%
yield. ESI-MS calculated for
C.sub.32H.sub.31N.sub.6O.sub.4[M+H].sup.+=563.24, Obtained:
563.45.
##STR00516##
Cpd. No. 287-TFA salt was prepared from amide condensation of
ZBA191 and 4-morpholinopiperidine using HBTU-HOBT condition. 67%
yield. ESI-MS calculated for
C.sub.35H.sub.36N.sub.7O.sub.4[M+H].sup.+=618.28, Obtained: 618.66.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.38 (d, J=5.1 Hz, 1H), 8.41
(d, J=8.5 Hz, 1H), 8.26 (d, J=5.1 Hz, 1H), 8.14 (dd, J=11.4, 4.1
Hz, 1H), 8.06 (d, J=8.5 Hz, 1H), 7.86-7.79 (m, 1H), 7.51 (s, 1H),
6.42 (s, 1H), 4.20-4.05 (m, 4H), 3.75 (t, J=12.1 Hz, 2H), 3.67-3.41
(m, 4H), 3.30 (s, 3H), 3.27-2.96 (m, 3H), 2.45-2.14 (m, 5H), 2.10
(s, 3H), 1.94-1.69 (m, 2H).
##STR00517##
Cpd. No. 288-TFA salt was prepared from Suzuki coupling of ZBA139
and CD223 using Pd(PPh.sub.3).sub.4-K.sub.2CO.sub.3 (2 M)
condition. 35% yield. ESI-MS calculated for
C.sub.26H.sub.21FN.sub.5O.sub.3 [M+H].sup.+=470.16, Obtained:
470.35. .sup.1H NMR (300 MHz, MeOD) .delta. 9.30 (s, 1H), 8.43 (dd,
J=9.2, 5.2 Hz, 1H), 8.10 (d, J=4.3 Hz, 1H), 7.86 (t, J=7.3 Hz, 1H),
7.65-7.55 (m, 2H), 6.31 (s, 1H), 5.12 (s, 2H), 3.99 (s, 3H), 2.29
(s, 3H), 2.10 (s, 3H).
##STR00518##
To a round-bottom flask, Cpd. No. 288 (0.045 g, 0.1 mmol) was
dissolved in DCM (7 mL) and Pyridine (0.4 mL) at room temperature.
Dess-martin periodinane (63.6 mg, 0.15 mmol) was added and the
reaction mixture was stirred for 2.5 h. Then water and ethyl
acetate was slowly added. The aqueous layer was extracted with
EtOAc. The combined EtOAc extracts were washed with H.sub.2O, dried
over Na.sub.2SO.sub.4, and concentrated under reduced pressure to
afford the aldehyde intermediate which then was dissolved in THF (3
mL), t-BuOH (3 mL) and H.sub.2O (1 mL) at room temperature.
NaClO.sub.2 (75 mg), NaH.sub.2PO.sub.4 (125 mg) and
2-Methyl-2-butene (0.5 mL) was added and the reaction mixture was
stirred overnight. Then water and ethyl acetate was slowly added.
The aqueous layer was extracted with EtOAc. The combined EtOAc
extracts were washed with H.sub.2O, dried over Na.sub.2SO.sub.4,
and concentrated under reduced pressure to afford Cpd. No. 289 (25
mg) after HPLC purification. ESI-MS calculated for
C.sub.26H.sub.19FN.sub.5O.sub.4 [M+H].sup.+=484.14, Obtained:
484.33.
##STR00519##
Cpd. No. 289 (20 mg), HBTU (24 mg), HOBt-H.sub.2O (6 mg) and DMF (1
mL) were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.05 mL)
was added followed by addition of
(S)-4-amino-1-(trityloxy)butan-2-ol (28 mg) was added and the
reaction mixture was stirred for 12 h. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
290 as a salt of CF.sub.3CO.sub.2H (15 mg). ESI-MS calculated for
C.sub.30H.sub.28FN.sub.6O.sub.5[M+H].sup.+=571.21, Obtained:
571.64. .sup.1H NMR (300 MHz, MeOD) .delta. 9.23 (d, J=4.6 Hz, 1H),
8.38 (dd, J=9.3, 5.2 Hz, 1H), 8.10 (d, J=4.6 Hz, 1H), 7.84-7.73 (m,
1H), 7.69-7.44 (m, 2H), 6.47 (s, 1H), 4.44-4.32 (m, 1H), 3.84-3.60
(m, 3H), 3.53 (d, J=5.5 Hz, 1H), 3.33 (s, 3H), 2.28 (s, 3H), 2.09
(s, 3H), 2.00-1.65 (m, 2H).
##STR00520##
Cpd. No. 291-TFA salt was prepared from amide condensation of Cpd.
No. 289 and 2-(4-aminopiperidin-1-yl)ethanol using HBTU-HOBT
condition. 50% yield. ESI-MS calculated for
C.sub.33H.sub.33FN.sub.7O.sub.4[M+H].sup.+=610.25, Obtained:
610.44. .sup.1H NMR (300 MHz, MeOD) .delta. 9.31 (d, J=5.1 Hz, 1H),
8.43 (dd, J=9.3, 5.0 Hz, 1H), 8.21 (d, J=5.0 Hz, 1H), 7.91-7.80 (m,
1H), 7.63-7.51 (m, 2H), 6.54 (s, 1H), 4.52-4.33 (m, 1H), 3.97-3.73
(m, 4H), 3.55-3.20 (m, 7H), 2.48-1.98 (m, 10H).
##STR00521##
Cpd. No. 292-TFA salt was prepared from amide condensation of Cpd.
No. 289 and 1-isopropyl-piperidin-4-ylamine using HBTU-HOBT
condition. 70% yield. ESI-MS calculated for
C.sub.34H.sub.35FN.sub.7O.sub.3[M+H].sup.+=608.27, Obtained:
608.66. .sup.1H NMR (300 MHz, MeOD) .delta. 9.23 (d, J=4.6 Hz, 1H),
8.38 (dd, J=9.3, 5.3 Hz, 1H), 8.05 (d, J=4.5 Hz, 1H), 7.79 (ddd,
J=9.3, 8.2, 2.8 Hz, 1H), 7.59 (s, 1H), 7.48 (dd, J=9.6, 2.7 Hz,
1H), 6.42 (s, 1H), 4.45-4.25 (m, 1H), 3.67-3.17 (m, 8H), 2.42-2.30
(m, 2H), 2.30 (s, 3H), 2.16-1.97 (m, 5H), 1.41 (d, J=6.7 Hz,
6H).
##STR00522##
Cpd. No. 293-TFA salt was prepared from amide condensation of Cpd.
No. 289 and 1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine using
HBTU-HOBT condition. 70% yield. ESI-MS calculated for
C.sub.36H.sub.37FN.sub.7O.sub.4[M+H].sup.+=650.28, Obtained:
650.46. .sup.1H NMR (300 MHz, MeOD) .delta. 9.26 (d, J=4.7 Hz, 1H),
8.39 (dd, J=9.4, 5.2 Hz, 1H), 8.10 (d, J=4.6 Hz, 1H), 7.82 (ddd,
J=9.3, 8.2, 2.8 Hz, 1H), 7.60 (s, 1H), 7.51 (dd, J=9.5, 2.6 Hz,
1H), 6.44 (s, 1H), 4.40-4.23 (m, 1H), 4.14-4.04 (m, 2H), 3.83-3.40
(m, 5H), 3.33 (s, 3H), 3.24 (t, J=12.2 Hz, 2H), 2.42-2.32 (m, 2H),
2.30 (s, 3H), 2.17-2.03 (m, 7H), 1.90-1.70 (m, 2H).
##STR00523##
Cpd. No. 294-TFA salt was prepared from amide condensation of Cpd.
No. 289 and 1-(oxetan-3-yl)piperidin-4-amine using HBTU-HOBT
condition. 40% yield. ESI-MS calculated for
C.sub.34H.sub.33FN.sub.7O.sub.4[M+H].sup.+=622.25, Obtained:
622.45. .sup.1H NMR (300 MHz, MeOD) .delta. 9.26 (d, J=4.7 Hz, 1H),
8.40 (dd, J=9.4, 5.2 Hz, 1H), 8.10 (d, J=4.7 Hz, 1H), 7.87-7.79 (m,
1H), 7.57 (s, 1H), 7.52 (dd, J=9.6, 2.7 Hz, 1H), 6.43 (s, 1H),
4.95-4.80 (m, 4H), 4.54-4.24 (m, 2H), 3.70-3.49 (m, 2H), 3.36 (s,
3H), 3.22-3.02 (m, 2H), 2.43-2.25 (m, 5H), 2.21-2.04 (m, 5H).
##STR00524##
methyl 8-bromo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1.1
g, 4.16 mmol), bis(pinacolato)diboron (2.13 g, 8.4 mmol, 2.0
equiv.), and potassium acetate (1.6 g, 16 mmol, 4.0 equiv.) were
added to a round-bottom flask Anhydrous 1,4-dixoane (20 mL) was
added via a syringe and the flask was degassed and refilled with
nitrogen. Pd(dppf)Cl.sub.2 (322 mg, 0.46 mmol, 0.1 equiv.) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to ambient
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound ZBA245 was isolated in
1.0 g. ESI-MS calculated for
C.sub.16H.sub.22BO.sub.6[M+H].sup.+=321.15, Obtained: 321.44.
##STR00525##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.4 g, 16 mmol, 1.0 equiv.) and ZBA245 (13.75 g, 37
mmol, 2.0 equiv.), 1,2-dimethoxyethane (150 mL), and
Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was degassed
to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.3 g, 1.6 mmol, 0.1 equiv.)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield the title compound
ZBA246 in 2.1 g (26.5% yield over two steps). ESI-MS calculated for
C.sub.27H.sub.25N.sub.4O.sub.6[M+H].sup.+=501.17, Obtained:
501.35.
##STR00526##
To a round-bottom flask, ZBA246 (110 mg, 0.22 mmol) was dissolved
in MeOH (5 mL) and water (5 mL). NaOH (26 mg, 0.66 mmol, 3 equiv.)
was added and solution was stirred for 3 h at 100.degree. C. The
reaction mixture was extracted with ethyl acetate. Subsequently,
the aqueous layer was neutralized to pH=2 and was extracted with
ethyl acetate. The organic extracts of acidic aqueous solution were
combined and concentrated on a rotary evaporator. The remaining
residue was freeze-dried to yield the title compound in 90 mg.
ESI-MS calculated for
C.sub.26H.sub.23N.sub.4O.sub.6[M+H].sup.+=487.16, Obtained:
487.35.
##STR00527##
(amide condensation): ZBA249 (20 mg, 0.05 mmol), EDCI-HCl (100 mg,
0.5 mmol), and HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a
round-bottom flask. EtN(i-Pr).sub.2 (0.1 mL) was added followed by
addition of DMF (2.5 mL). N,N-dimethylethylenediamine (40 mg) was
added and the reaction mixture was stirred for 12 h. The reaction
was quenched with NaHCO.sub.3 saturated solution and the aqueous
layer was extracted with ethyl acetate. The combined organic layers
were concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the title compound Cpd.
No. 295 as a salt of CF.sub.3CO.sub.2H (69% yield). ESI-MS
calculated for C.sub.30H.sub.33N.sub.6O.sub.5[M+H].sup.+=557.25,
Obtained: 557.44. .sup.1H NMR (300 MHz, MeOD) .delta. 7.85 (d,
J=8.2 Hz, 1H), 7.58 (s, 1H), 7.50 (d, J=8.2 Hz, 1H), 7.19 (s, 1H),
4.61-4.54 (m, 2H), 4.45-4.40 (m, 2H), 3.89 (t, J=5.9 Hz, 2H), 3.71
(s, 3H), 3.47 (t, J=5.9 Hz, 2H), 3.04 (s, 6H), 2.98 (s, 3H), 2.33
(s, 3H), 2.15 (s, 3H).
##STR00528##
ZBA249 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). 1-Boc-piperazine (40 mg) was added and the reaction mixture
was stirred for 12 h. The reaction was quenched with NaHCO.sub.3
saturated solution and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were concentrated on a rotary
evaporator. The remaining residue was dissolved in TFA (2 mL) and
DCM (2 mL). The mixture was stirred for 3 hours and was
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 296 as a salt
of CF.sub.3CO.sub.2H (15 mg). ESI-MS calculated for
C.sub.30H.sub.31N.sub.6O.sub.5[M+H].sup.+=555.23, Obtained: 555.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.58 (s, 1H), 7.51 (d, J=7.9
Hz, 1H), 7.29 (d, J=7.9 Hz, 1H), 7.21 (s, 1H), 4.59-4.37 (m, 4H),
4.23-3.96 (m, 2H), 3.82-3.62 (m, 5H), 3.51-3.30 (m, 4H), 2.99 (s,
3H), 2.33 (s, 3H), 2.15 (s, 3H).
##STR00529##
Cpd. No. 297-TFA salt was prepared from amide condensation of
ZBA249 and 1-methylpiperazine using EDCI-HOBT condition. 75% yield.
ESI-MS calculated for
C.sub.31H.sub.33N.sub.6O.sub.5[M+H].sup.+=569.25, Obtained: 569.64.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.56 (s, 1H), 7.50 (d, J=7.9
Hz, 1H), 7.29 (d, J=7.9 Hz, 1H), 7.19 (s, 1H), 4.55-4.37 (m, 4H),
3.92-3.32 (m, 11H), 3.02 (s, 3H), 2.98 (s, 3H), 2.34 (s, 3H), 2.16
(s, 3H).
##STR00530##
ZBA249 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). (S)-1-N-Boc-2-methylpiperazine (40 mg) was added and the
reaction mixture was stirred for 12 h. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
298 as a salt of CF.sub.3CO.sub.2H (17 mg). ESI-MS calculated for
C.sub.31H.sub.33N.sub.6O.sub.5[M+H].sup.+=569.25, Obtained:
569.55.
##STR00531##
Cpd. No. 299-TFA salt was prepared from amide condensation of
ZBA249 and (S)-1,2-dimethylpiperazine dihydrochloride using
EDCI-HOBT condition. 75% yield. ESI-MS calculated for
C.sub.32H.sub.35N.sub.6O.sub.5[M+H].sup.+=583.26, Obtained: 583.37.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.58 (s, 1H), 7.51 (d, J=7.9
Hz, 1H), 7.33-7.18 (m, 2H), 4.60-4.35 (m, 4H), 3.96-3.31 (m, 10H),
3.06-2.96 (m, 6H), 2.34 (s, 3H), 2.16 (s, 3H), 1.59-1.32 (m,
3H).
##STR00532##
Cpd. No. 300-TFA salt was prepared from amide condensation of
ZBA249 and cis-2,6-dimethylpiperazine dihydrochloride using
EDCI-HOBT condition. 80% yield. ESI-MS calculated for
C.sub.32H.sub.35N.sub.6O.sub.5[M+H].sup.+=583.26, Obtained: 583.47.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.58 (s, 1H), 7.51 (d, J=5.5
Hz, 1H), 7.36-7.18 (m, 2H), 4.91 (brs, 1H), 4.55-4.36 (m, 4H),
3.88-3.69 (m, 4H), 3.63-3.20 (m, 3H), 3.06-2.86 (m, 4H), 2.34 (s,
3H), 2.16 (s, 3H), 1.47 (d, J=6.5 Hz, 3H), 1.40-1.27 (m, 3H).
##STR00533##
Cpd. No. 301-TFA salt was prepared from amide condensation of
ZBA249 and 1-(2-hydroxyethyl)piperazine using EDCI-HOBT condition.
80% yield. ESI-MS calculated for
C.sub.32H.sub.35N.sub.6O.sub.6[M+H].sup.+=599.26, Obtained: 599.66.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.57 (s, 1H), 7.51 (d, J=7.9
Hz, 1H), 7.29 (d, J=7.9 Hz, 1H), 7.21 (s, 1H), 4.59-4.37 (m, 4H),
4.01-3.92 (m, 2H), 3.89-3.35 (m, 13H), 2.99 (s, 3H), 2.34 (s, 3H),
2.16 (s, 3H).
##STR00534##
Cpd. No. 302-TFA salt was prepared from amide condensation of
ZBA249 and 1-methyl-4-piperidinamine using EDCI-HOBT condition. 75%
yield. ESI-MS calculated for
C.sub.32H.sub.35N.sub.6O.sub.5[M+H].sup.+=583.26, Obtained: 583.37.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.64 (d, J=8.1 Hz, 1H), 7.58
(s, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.19 (s, 1H), 4.63-4.33 (m, 4H),
4.37-4.14 (m, 1H), 3.72 (s, 3H), 3.70-3.50 (m, 2H), 3.22 (dd,
J=13.2, 10.6 Hz, 2H), 2.98 (s, 3H), 2.93 (s, 3H), 2.38-2.24 (d,
J=8.3 Hz, 5H), 2.16 (s, 3H), 2.08-1.90 (m, 2H).
##STR00535##
ZBA249 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). 4-Amino-1-Boc-piperidine (40 mg) was added and the reaction
mixture was stirred for 12 h. The reaction was quenched with
NaHCO.sub.3 saturated solution and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were concentrated
on a rotary evaporator. The remaining residue was dissolved in TFA
(2 mL) and DCM (2 mL). The mixture was stirred for 3 hours and was
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 303 as a salt
of CF.sub.3CO.sub.2H (20 mg). ESI-MS calculated for
C.sub.31H.sub.33N.sub.6O.sub.5[M+H].sup.+=569.25, Obtained: 569.45.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.64 (d, J=8.1 Hz, 1H), 7.58
(s, 1H), 7.48 (d, J=8.1 Hz, 1H), 7.19 (s, 1H), 4.63-4.48 (m, 2H),
4.44-4.38 (m, 2H), 4.34-4.17 (m, 1H), 3.72 (s, 3H), 3.52 (dd,
J=9.7, 3.6 Hz, 2H), 3.22 (td, J=12.7, 2.8 Hz, 2H), 2.98 (s, 3H),
2.37-2.23 (m, 5H), 2.16 (s, 3H), 2.02-1.85 (m, 2H).
##STR00536##
Cpd. No. 304-TFA salt was prepared from amide condensation of
ZBA249 and 2-(4-aminopiperidin-1-yl)ethanol using EDCI-HOBT
condition. 75% yield. ESI-MS calculated for
C.sub.33H.sub.37N.sub.6O.sub.6[M+H].sup.+=613.27, Obtained: 613.57.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.64 (d, J=8.1 Hz, 1H), 7.58
(s, 1H), 7.48 (d, J=8.1 Hz, 1H), 7.19 (s, 1H), 4.64-4.17 (m, 5H),
3.98-3.88 (m, 2H), 3.85-3.66 (m, 5H), 3.62-3.36 (m, 2H), 3.30-3.15
(m, 2H), 2.98 (s, 3H), 2.41-1.90 (m, 10H).
##STR00537##
Cpd. No. 305-TFA salt was prepared from amide condensation of
ZBA249 and 1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine using
EDCI-HOBT condition. 80% yield. ESI-MS calculated for
C.sub.36H.sub.41N.sub.6O.sub.6[M+H].sup.+=653.30, Obtained: 653.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.64 (d, J=8.1 Hz, 1H), 7.58
(s, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.19 (s, 1H), 4.62-4.35 (m, 4H),
4.32-4.18 (m, 1H), 4.11 (dd, J=11.3, 4.1 Hz, 2H), 3.78-3.66 (m,
5H), 3.48 (t, J=11.3 Hz, 3H), 3.30-3.17 (m, 2H), 2.98 (s, 3H),
2.43-2.26 (m, 5H), 2.16 (s, 3H), 2.13-1.70 (m, 6H).
##STR00538##
Cpd. No. 306-TFA salt was prepared from amide condensation of
ZBA249 and 1-(oxetan-3-yl)piperidin-4-amine using EDCI-HOBT
condition. 80% yield. ESI-MS calculated for
C.sub.34H.sub.37N.sub.6O.sub.6[M+H].sup.+=625.27, Obtained: 625.37.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.63 (d, J=8.1 Hz, 1H), 7.58
(s, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.19 (s, 1H), 4.89 (d, J=6.5 Hz,
4H), 4.63-4.18 (m, 6H), 3.72 (s, 3H), 3.65-3.45 (m, 2H), 3.22-3.02
(m, 2H), 2.98 (s, 3H), 2.45-1.91 (m, 10H).
##STR00539##
Cpd. No. 307-TFA salt was prepared from amide condensation of
ZBA249 and 1-(oxetan-3-yl)piperazine using EDCI-HOBT condition. 70%
yield. ESI-MS calculated for
C.sub.33H.sub.35N.sub.6O.sub.6[M+H].sup.+=611.26, Obtained: 611.37.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.58 (s, 1H), 7.51 (d, J=7.9
Hz, 1H), 7.29 (d, J=7.9 Hz, 1H), 7.19 (d, J=10.4 Hz, 1H), 4.95-4.87
(m, 4H), 4.60-4.34 (m, 5H), 4.34-3.76 (m, 4H), 3.73 (s, 3H),
3.50-3.20 (m, 4H), 2.99 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H).
##STR00540##
Cpd. No. 308-TFA salt was prepared from amide condensation of
ZBA249 and 1-methylsulfonyl-piperazine using EDCI-HOBT condition.
80% yield. ESI-MS calculated for
C.sub.31H.sub.33N.sub.6O.sub.7S[M+H].sup.+=633.21, Obtained:
633.44. .sup.1H NMR (300 MHz, MeOD) .delta. 7.59 (s, 1H), 7.51 (d,
J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.22 (s, 1H), 4.59-4.38 (m,
4H), 3.98-3.88 (m, 2H), 3.74 (s, 3H), 3.66-3.47 (m, 2H), 3.43-3.25
(m, 4H), 3.00 (s, 3H), 2.92 (s, 3H), 2.33 (s, 3H), 2.15 (s,
3H).
##STR00541##
ZBA249 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). (S)-4-Amino-1-(trityloxy)butan-2-ol (40 mg) was added and the
reaction mixture was stirred for 12 h. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
309 as a salt of CF.sub.3CO.sub.2H (18 mg). ESI-MS calculated for
C.sub.30H.sub.32N.sub.5O.sub.7[M+H].sup.+=574.23, Obtained: 574.47.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.77 (d, J=8.2 Hz, 1H), 7.57
(s, 1H), 7.47 (d, J=8.2 Hz, 1H), 7.18 (s, 1H), 4.61-4.51 (m, 2H),
4.45-4.37 (m, 2H), 3.85-3.52 (m, 8H), 2.98 (s, 3H), 2.34 (s, 3H),
2.16 (s, 3H), 2.00-1.61 (m, 2H).
##STR00542##
Cpd. No. 310-TFA salt was prepared from amide condensation of
ZBA249 and 4-hydroxypiperidine using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for C.sub.31H.sub.32N.sub.5O.sub.6
[M+H].sup.+=570.23, Obtained: 570.4. .sup.1H NMR (300 MHz, MeOD)
.delta. 7.57 (s, 1H), 7.47 (dd, J=7.9, 3.6 Hz, 1H), 7.26-7.15 (m,
2H), 4.54-4.35 (m, 4H), 4.32-4.18 (m, 1H), 4.02-3.90 (m, 1H), 3.73
(s, 3H), 3.70-3.55 (m, 1H), 3.51-3.21 (m, 2H), 2.99 (s, 3H), 2.34
(s, 3H), 2.16 (s, 3H), 2.07-1.78 (m, 2H), 1.68-1.4256 (m, 2H).
##STR00543##
Cpd. No. 311-TFA salt was prepared from amide condensation of
ZBA249 and cis-4-Amino-cyclohexanol using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for C.sub.32H.sub.34N.sub.5O.sub.6
[M+H].sup.+=584.25, Obtained: 584.5.
##STR00544##
Cpd. No. 312-TFA salt was prepared from amide condensation of
ZBA249 and morpholine using EDCI-HOBT condition. 80% yield. ESI-MS
calculated for C.sub.30H.sub.30N.sub.5O.sub.6 [M+H].sup.+=556.21,
Obtained: 556.4. .sup.1H NMR (300 MHz, MeOD) .delta. 7.57 (s, 1H),
7.48 (d, J=7.9 Hz, 1H), 7.24 (d, J=7.9 Hz, 1H), 7.18 (s, 1H),
4.51-4.45 (m, 2H), 4.44-4.36 (m, 2H), 3.88-3.66 (m, 9H), 3.52-3.39
(m, 2H), 2.98 (s, 3H), 2.34 (s, 3H), 2.16 (s, 3H).
##STR00545##
Cpd. No. 313-TFA salt was prepared from amide condensation of
ZBA249 and 4-morpholinopiperidine using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for C.sub.35H.sub.39N.sub.6O.sub.6
[M+H].sup.+=639.29, Obtained: 639.5. .sup.1H NMR (300 MHz, MeOD)
.delta. 7.57 (s, 1H), 7.49 (d, J=7.9 Hz, 1H), 7.32-7.17 (m, 2H),
4.59-4.28 (m, 4H), 4.20-4.02 (m, 2H), 3.93-3.15 (m, 13H), 3.05-2.86
(m, 4H), 2.46-2.22 (m, 5H), 2.16 (s, 3H), 1.94-1.66 (m, 2H).
##STR00546##
1-methyl-4-piperidinecarboxylic acid (57 mg, 0.4 mmol) and THF (10
mL) were added to a round-bottom flask. Oxalyl chloride (0.1 mL)
was added followed by addition of DMF (1 drop). The reaction
mixture was stirred for 3 h and then was concentrated on a rotary
evaporator. The remaining residue was dissolved in DMF (1 mL). Then
Cpd. No. 150 (20 mg) and NaHCO.sub.3 (50 mg) was added. The mixture
was stirred for 12 h at room temperature. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 314 as a salt
of CF.sub.3CO.sub.2H (13 mg). ESI-MS calculated for
C.sub.34H.sub.35N.sub.6O.sub.3[M+H].sup.+=575.27, Obtained: 575.47.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.38 (d, J=8.4 Hz, 1H), 8.13
(d, J=7.8 Hz, 1H), 8.04 (d, J=7.8 Hz, 1H), 7.79 (t, J=8.7 Hz, 2H),
7.67-7.60 (m, 1H), 7.55 (s, 1H), 6.23 (s, 1H), 3.77-3.66 (m, 2H),
3.54 (d, J=7.3 Hz, 2H), 3.27-3.09 (m, 4H), 3.03 (s, 3H), 2.97 (s,
3H), 2.47-2.12 (m, 7H), 2.08 (s, 3H).
##STR00547##
Cpd. No. 150 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-methyl-4-piperidinamine (300 mg) and DBU (300 mg) was added. The
mixture was stirred for 12 h at room temperature. The reaction was
quenched with NaHCO.sub.3 saturated solution and the aqueous layer
was extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 315 as a salt
of CF.sub.3CO.sub.2H (30 mg). ESI-MS calculated for
C.sub.34H.sub.36N.sub.7O.sub.3[M+H].sup.+=590.28 Obtained: 590.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.51-8.31 (m, 2H), 7.98 (d,
J=8.1 Hz, 1H), 7.82-7.71 (m, 2H), 7.65-7.58 (m, 1H), 7.54 (s, 1H),
6.26 (s, 1H), 4.06-3.90 (m, 1H), 3.71-3.47 (m, 2H), 3.28-3.12 (m,
5H), 3.02 (s, 3H), 2.93 (s, 3H), 2.44-2.14 (m, 5H), 2.09 (s, 3H),
1.95-1.77 (m, 2H).
##STR00548##
Cpd. No. 150 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-methylpiperazine (300 mg) and DBU (300 mg) was added. The mixture
was stirred for 12 h at room temperature. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 316 as a salt
of CF.sub.3CO.sub.2H (29 mg). ESI-MS calculated for
C.sub.33H.sub.34N.sub.7O.sub.3[M+H].sup.+=576.27, Obtained: 576.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.34 (d, J=8.5 Hz, 1H), 8.02
(d, J=7.8 Hz, 1H), 7.87 (d, J=7.8 Hz, 1H), 7.81-7.69 (m, 2H),
7.65-7.56 (m, 1H), 7.55 (s, 1H), 6.26 (s, 1H), 4.70-4.34 (m, 2H),
3.81-3.24 (m, 6H), 3.20 (s, 3H), 3.04 (s, 3H), 3.03 (s, 3H), 2.28
(s, 3H), 2.09 (s, 3H).
##STR00549##
Cpd. No. 150 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-(oxetan-3-yl)piperazine (300 mg) and DBU (300 mg) was added. The
mixture was stirred for 12 h at room temperature. The reaction was
quenched with NaHCO.sub.3 saturated solution and the aqueous layer
was extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 317 as a salt
of CF.sub.3CO.sub.2H (32 mg). ESI-MS calculated for
C.sub.35H.sub.36N.sub.7O.sub.4[M+H].sup.+=618.28, Obtained: 618.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.33 (d, J=8.5 Hz, 1H), 8.01
(d, J=7.8 Hz, 1H), 7.87 (d, J=7.8 Hz, 1H), 7.82-7.69 (m, 2H),
7.66-7.57 (m, 1H), 7.55 (s, 1H), 6.26 (s, 1H), 5.01-4.84 (m, 4H),
4.57-4.46 (m, 1H), 4.13-3.97 (m, 4H), 3.44-3.36 (m, 4H), 3.19 (s,
3H), 3.03 (s, 3H), 2.28 (s, 3H), 2.08 (s, 3H).
##STR00550##
Cpd. No. 150 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-(tetrahydro-2H-pyran-4-yl)piperazine (300 mg) and DBU (300 mg)
was added. The mixture was stirred for 12 h at room temperature.
The reaction was quenched with NaHCO.sub.3 saturated solution and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were concentrated on a rotary evaporator. The
remaining residue was purified by reverse phase HPLC affording the
Cpd. No. 318 as a salt of CF.sub.3CO.sub.2H (5 mg). ESI-MS
calculated for C.sub.37H.sub.40N.sub.7O.sub.4[M+H].sup.+=646.31,
Obtained: 646.55.
##STR00551##
Methyl 3-bromo-5-(tert-butyl)benzoate (1.1 g, 4.16 mmol),
bis(pinacolato)diboron (2.13 g, 8.4 mmol, 2.0 equiv.), and
potassium acetate (1.6 g, 16 mmol, 4.0 equiv.) were added to a
round-bottom flask Anhydrous 1,4-dixoane (20 mL) was added via a
syringe and the flask was degassed and refilled with nitrogen.
Pd(dppf)Cl.sub.2 (322 mg, 0.46 mmol, 0.1 equiv.) was added and the
system was degassed again followed by heating at 100.degree. C. for
16 h. The reaction mixture was cooled to ambient temperature and
diluted by CH.sub.2Cl.sub.2. The solution was filtered through a
pad of celite and the volatile components were removed on a rotary
evaporator. The residue was purified by flash column
chromatography. The title compound ZBA297 was isolated in 0.9 g.
ESI-MS calculated for C.sub.18H.sub.28BO.sub.4[M+H].sup.+=319.20,
Obtained: 319.4.
##STR00552##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.4 g, 16 mmol, 1.0 equiv.) and ZBA297 (13.75 g, 37
mmol, 2.0 equiv.), 1,2-dimethoxyethane (150 mL), and
Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was degassed
to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.3 g, 1.6 mmol, 0.1 equiv.)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield the title compound
ZBA298 in 2.2 g. ESI-MS calculated for
C.sub.29H.sub.31N.sub.4O.sub.4[M+H].sup.+=499.23, Obtained: 499.6.
.sup.1H NMR (300 MHz, DMSO) .delta. 12.28 (s, 1H), 8.37 (s, 1H),
8.22 (s, 2H), 7.40 (s, 1H), 7.31 (s, 1H), 3.91 (s, 3H), 3.62 (s,
3H), 2.76 (s, 3H), 2.30 (s, 3H), 2.09 (s, 3H), 1.42 (s, 9H).
##STR00553##
To a round-bottom flask, ZBA298 (110 mg, 0.22 mmol) was dissolved
in MeOH (5 mL) and water (5 mL). NaOH (26 mg, 0.66 mmol, 3 equiv.)
was added and solution was stirred for 3 h at 100.degree. C. The
reaction mixture was extracted with ethyl acetate. Subsequently,
the aqueous layer was neutralized to pH=2 and was extracted with
ethyl acetate. The organic extracts of acidic aqueous solution were
combined and concentrated on a rotary evaporator. The remaining
residue was freeze-dried to yield the title compound in 80 mg.
ESI-MS calculated for
C.sub.28H.sub.29N.sub.4O.sub.4[M+H].sup.+=485.21, Obtained: 485.5.
.sup.1H NMR (300 MHz, DMSO) .delta. 13.34 (brs, 1H), 12.56 (brs,
1H), 8.40 (t, J=1.4 Hz, 1H), 8.30-8.18 (m, 2H), 7.44 (s, 1H), 7.32
(s, 1H), 3.63 (s, 3H), 2.80 (s, 3H), 2.30 (s, 3H), 2.09 (s, 3H),
1.42 (s, 9H).
##STR00554##
Cpd. No. 319-TFA salt was prepared from amide condensation of
ZBA301 and 1-methyl-4-piperidinamine using EDCI-HOBT condition. 75%
yield. ESI-MS calculated for
C.sub.34H.sub.41N.sub.6O.sub.3[M+H].sup.+=581.32, Obtained: 581.66.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.42 (t, J=1.6 Hz, 1H), 8.38
(t, J=1.7 Hz, 1H), 8.23 (t, J=1.7 Hz, 1H), 7.59 (s, 1H), 7.29 (s,
1H), 4.38-4.17 (m, 1H), 3.74-3.57 (m, 5H), 3.21 (dd, J=13.2, 10.6
Hz, 2H), 3.00 (s, 3H), 2.92 (s, 3H), 2.35-2.22 (m, 5H), 2.15 (s,
3H), 2.10-1.88 (m, 2H), 1.52 (s, 9H).
##STR00555##
Cpd. No. 320-TFA salt was prepared from amide condensation of
ZBA301 and 1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine using
EDCI-HOBT condition. 80% yield. ESI-MS calculated for
C.sub.38H.sub.47N.sub.6O.sub.4[M+H].sup.+=651.36, Obtained: 651.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.42 (t, J=1.6 Hz, 1H), 8.39
(t, J=1.6 Hz, 1H), 8.23 (t, J=1.7 Hz, 1H), 7.59 (s, 1H), 7.30 (s,
1H), 4.35-4.20 (m, 1H), 4.15-4.05 (m, 2H), 3.80-3.65 (m, 5H),
3.55-3.40 (m, 3H), 3.22 (t, J=12.0 Hz, 2H), 3.00 (s, 3H), 2.40-2.20
(m, 5H), 2.15 (s, 3H), 2.12-1.69 (m, 6H), 1.52 (s, 9H).
##STR00556##
Cpd. No. 321-TFA salt was prepared from amide condensation of
ZBA301 and 1-(oxetan-3-yl)piperazine using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for
C.sub.35H.sub.41N.sub.6O.sub.4[M+H].sup.+=609.31, Obtained: 609.46.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.18 (t, J=1.7 Hz, 1H), 8.01
(s, 1H), 8.01 (s, 1H), 7.58 (s, 1H), 7.29 (s, 1H), 4.88 (d, J=6.4
Hz, 4H), 4.44 (p, J=6.3 Hz, 1H), 4.22-3.78 (m, 4H), 3.74 (s, 3H),
3.43-3.24 (m, 4H), 3.00 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H), 1.50
(s, 9H).
##STR00557##
Cpd. No. 322-TFA salt was prepared from amide condensation of
ZBA301 and 1-(oxetan-3-yl)piperidin-4-amine using EDCI-HOBT
condition. 90% yield. ESI-MS calculated for
C.sub.36H.sub.43N.sub.6O.sub.4[M+H].sup.+=623.33, Obtained: 623.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.42 (t, J=1.5 Hz, 1H), 8.39
(t, J=1.6 Hz, 1H), 8.22 (t, J=1.6 Hz, 1H), 7.59 (s, 1H), 7.28 (s,
1H), 4.88 (d, J=6.5 Hz, 4H), 4.53-4.40 (m, 1H), 4.35-4.20 (m, 1H),
3.68 (s, 3H), 3.65-3.50 (m, 2H), 3.25-3.02 (m, 2H), 3.00 (s, 3H),
2.35-2.25 (m, 5H), 2.20-2.00 (m, 5H), 1.51 (s, 9H).
##STR00558##
Cpd. No. 323-TFA salt was prepared from amide condensation of
ZBA301 and 1-methylpiperazine using EDCI-HOBT condition. 70% yield.
ESI-MS calculated for
C.sub.33H.sub.39N.sub.6O.sub.3[M+H].sup.+=567.30, Obtained: 567.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.18 (t, J=1.7 Hz, 1H), 8.01
(s, 1H), 8.00 (s, 1H), 7.58 (s, 1H), 7.30 (s, 1H), 3.74 (s, 3H),
3.68-3.16 (m, 8H), 3.00 (s, 3H), 2.97 (s, 3H), 2.33 (s, 3H), 2.15
(s, 3H), 1.51 (s, 9H).
##STR00559##
Cpd. No. 324-TFA salt was prepared from amide condensation of
ZBA301 and 1-isopropylpiperazine using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for
C.sub.35H.sub.43N.sub.6O.sub.3[M+H].sup.+=595.33, Obtained: 595.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.18 (t, J=1.7 Hz, 1H), 8.02
(s, 1H), 8.02 (s, 1H), 7.58 (s, 1H), 7.29 (s, 1H), 3.74 (s, 3H),
3.68-3.15 (m, 9H), 3.00 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H), 1.51
(s, 9H), 1.40 (d, J=6.6 Hz, 6H).
##STR00560##
Cpd. No. 325-TFA salt was prepared from amide condensation of
ZBA301 and 1-(2-hydroxyethyl)piperazine using EDCI-HOBT condition.
50% yield. ESI-MS calculated for
C.sub.34H.sub.41N.sub.6O.sub.4[M+H].sup.+=597.31, Obtained: 597.5.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.17 (t, J=1.7 Hz, 1H),
8.00-7.97 (m, 2H), 7.56 (s, 1H), 7.30 (s, 1H), 4.00-3.80 (m, 4H),
3.74 (s, 3H), 3.68-3.18 (m, 8H), 2.98 (s, 3H), 2.33 (s, 3H), 2.15
(s, 3H), 1.51 (s, 9H).
##STR00561##
Cpd. No. 162 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-methylpiperazine (300 mg) and DBU (300 mg) was added. The mixture
was stirred for 12 h at room temperature. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 326 as a salt
of CF.sub.3CO.sub.2H (5 mg). ESI-MS calculated for
C.sub.33H.sub.40N.sub.7O.sub.3[M+H].sup.+=582.31, Obtained: 582.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.85 (d, J=2.1 Hz, 1H),
7.76-7.69 (m, 2H), 7.61 (s, 1H), 7.05 (d, J=8.4 Hz, 1H), 4.27-3.25
(m, 11H), 3.05 (s, 3H), 2.98 (s, 3H), 2.35 (s, 3H), 2.17 (s, 3H),
1.52 (s, 9H).
##STR00562##
Cpd. No. 162 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-(tetrahydro-2H-pyran-4-yl)piperazine (300 mg) and DBU (300 mg)
was added. The mixture was stirred for 12 h at room temperature.
The reaction was quenched with NaHCO.sub.3 saturated solution and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were concentrated on a rotary evaporator. The
remaining residue was purified by reverse phase HPLC affording the
Cpd. No. 327 as a salt of CF.sub.3CO.sub.2H (25 mg). ESI-MS
calculated for C.sub.37H.sub.46N.sub.7O.sub.4[M+H].sup.+=652.36,
Obtained: 652.45. .sup.1H NMR (300 MHz, MeOD) .delta. 7.86 (d,
J=2.1 Hz, 1H), 7.76-7.71 (m, 2H), 7.61 (s, 1H), 7.07 (d, J=8.4 Hz,
1H), 4.39-3.39 (m, 16H), 2.99 (s, 3H), 2.35 (s, 3H), 2.20-2.08 (m,
5H), 1.99-1.69 (m, 2H), 1.52 (s, 9H).
##STR00563##
Cpd. No. 162 (50 mg) and pyridine (2 mL) were added to a
round-bottom flask. 4-Nitrophenyl chloroformate (33 mg) was added.
The reaction mixture was stirred for 5 h and then
1-(oxetan-3-yl)piperazine (300 mg) and DBU (300 mg) was added. The
mixture was stirred for 12 h at room temperature. The reaction was
quenched with NaHCO.sub.3 saturated solution and the aqueous layer
was extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
purified by reverse phase HPLC affording the Cpd. No. 328 as a salt
of CF.sub.3CO.sub.2H (34 mg). ESI-MS calculated for
C.sub.35H.sub.42N.sub.7O.sub.4[M+H].sup.+=624.32, Obtained: 624.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.86 (d, J=2.1 Hz, 1H),
7.76-7.71 (m, 2H), 7.61 (s, 1H), 7.06 (d, J=8.4 Hz, 1H), 4.91-4.81
(m, 4H), 4.47-4.33 (m, 1H), 4.30-3.10 (m, 11H), 2.99 (s, 3H), 2.35
(s, 3H), 2.16 (s, 3H), 1.52 (s, 9H).
##STR00564##
Isobutyl aldehyde (0.7 mL), 2-oxopiperazine (0.5 g) and
trans-1,2-Bis(phenylsulfonyl)ethylene (1.7 g) were mixture in a
round-bottom flask followed by addition of anhydrous PhMe (100 mL)
and 4A molecular sieve (1 g). The reaction mixture was stirred at
80.degree. C. overnight. The solid was filter off and the solution
was concentrated on a rotary evaporator. The remaining residue was
dissolved in THF (20 mL) and DBU (1.5 mL) was added. The mixture
was stirred at room temperature for 4 h. The mixture was diluted
with ethyl acetate and wash with 1N HCl to remove DBU. The organic
layer was dried, concentrated on a rotary evaporator. The remaining
residue was purified by flash column chromatography to yield ZBA310
in 450 mg. ESI-MS calculated for C.sub.10H.sub.15N.sub.2O
[M+H].sup.+=179.11, Obtained: 179.33.
##STR00565##
ZBA310 (350 mg) was dissolved in CHCl.sub.3 (20 mL). NBS (350 mg)
was added in small portions and the mixture was stirred at room
temperature for 2 h. The volatile components were removed on a
rotary evaporator. The remaining residue was purified by flash
column chromatography to yield ZBB12 in 280 mg. ESI-MS calculated
for C.sub.10H.sub.14BrN.sub.2O [M+H].sup.+=257.02, Obtained:
257.15.
##STR00566##
ZBB12 (250 mg) was dissolved in DMF (2 mL). NaH (40 mg) was added
in small portions and then MeI (0.1 mL) was added. The mixture was
stirred at room temperature for 2 h. The mixture was diluted with
ethyl acetate and wash with aq. NaCl. The organic layer was dried,
concentrated on a rotary evaporator. The remaining residue was
purified by flash column chromatography to yield ZBB15 in 230 mg.
ESI-MS calculated for C.sub.11H.sub.16N.sub.2OBr
[M+H].sup.+=271.04, Obtained: 271.32.
##STR00567##
ZBB15 (250 mg) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.4 mL) were
dissolved in anhydrous THF (20 mL). The solution was cooled to
-78.degree. C. for 15 min before BuLi (0.77 mL, 2.5 M in THF) was
added via a syringe. The reaction was stirred at -78.degree. C. for
6 h before quenching with saturated NH.sub.4Cl aqueous solution.
The aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine, dried over anhydrous sodium
sulfate, and concentrated on a rotary evaporator. The remaining
residue was purified by flash column chromatography to yield the
title compound ZBB19 in 180 mg. ESI-MS calculated for
C.sub.17H.sub.28N.sub.2O.sub.3B[M+H].sup.+=319.21, Obtained:
319.44.
##STR00568##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 70 mg) and ZBB19 (130 mg), 1,2-dimethoxyethane (150
mL), and Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was
degassed to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (25 mg) was added and the system
was degassed and refilled with nitrogen. The reaction mixture was
heated at reflux for 16 h. The reaction was quenched with water and
the aqueous layer was extracted with ethyl acetate. The organic
layers were combined and the volatile components were removed on a
rotary evaporator. The residue was purified by flash column
chromatography to yield the title compound Cpd. No. 329 in 15 mg.
ESI-MS calculated for
C.sub.28H.sub.31N.sub.6O.sub.3[M+H].sup.+=499.24, Obtained: 499.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 7.56 (s, 1H), 7.17 (s, 1H),
7.10 (s, 1H), 4.53-4.41 (m, 2H), 3.95-3.83 (m, 2H), 3.71 (s, 3H),
3.30-3.20 (m, 1H), 3.19 (s, 3H), 2.95 (s, 3H), 2.33 (s, 3H), 2.15
(s, 3H), 1.24 (d, J=7.2 Hz, 6H).
##STR00569##
Cpd. No. 329 (8 mg) and LiAlH.sub.4 (4 mg) were dissolved in
anhydrous THF (5 mL). The solution was heated to 70.degree. C. for
3 hours before quenching with saturated NH.sub.4Cl aqueous
solution. The aqueous layer was extracted with ethyl acetate and
the combined organic layers were washed with brine, dried over
anhydrous sodium sulfate, and concentrated on a rotary evaporator.
The remaining residue was purified by reverse phase HPLC affording
the Cpd. NO. 330 as a salt of CF.sub.3CO.sub.2H (5 mg). ESI-MS
calculated for C.sub.28H.sub.33N.sub.6O.sub.2[M+H].sup.+=485.26,
Obtained: 485.44. .sup.1H NMR (300 MHz, MeOD) .delta. 7.56 (s, 1H),
7.17 (s, 1H), 6.49 (s, 1H), 4.65 (brs, 2H), 4.55 (t, J=5.6 Hz, 2H),
3.92 (brs, 2H), 3.75 (s, 3H), 3.30-3.21 (m, 1H), 3.16 (s, 3H), 2.94
(s, 3H), 2.34 (s, 3H), 2.16 (s, 3H), 1.23 (d, J=7.2 Hz, 6H).
##STR00570##
Methyl 5-bromoquinoline-8-carboxylate (1.1 g, 4.16 mmol),
bis(pinacolato)diboron (2.13 g, 8.4 mmol, 2.0 equiv.), and
potassium acetate (1.6 g, 16 mmol, 4.0 equiv.) were added to a
round-bottom flask Anhydrous 1,4-dixoane (20 mL) was added via a
syringe and the flask was degassed and refilled with nitrogen.
Pd(dppf)Cl.sub.2 (322 mg, 0.46 mmol, 0.1 equiv.) was added and the
system was degassed again followed by heating at 100.degree. C. for
16 h. The reaction mixture was cooled to ambient temperature and
diluted by CH.sub.2Cl.sub.2. The solution was filtered through a
pad of celite and the volatile components were removed on a rotary
evaporator. The residue was purified by flash column
chromatography. The title compound ZBB23 was isolated in 0.7 g.
ESI-MS calculated for C.sub.17H.sub.21BNO.sub.4[M+H].sup.+=314.15,
Obtained: 314.33.
##STR00571##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.4 g, 16 mmol, 1.0 equiv.) and ZBB23 (18.2 g, 37
mmol, 2.0 equiv.), 1,2-dimethoxyethane (150 mL), and
Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was degassed
to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.3 g, 1.6 mmol, 0.1 equiv.)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield the title compound
ZBB25 in 3.7 g. ESI-MS calculated for
C.sub.28H.sub.24N.sub.5O.sub.4[M+H].sup.+=494.18, Obtained:
494.33.
##STR00572##
To a round-bottom flask, ZBB25 (110 mg, 0.22 mmol) was dissolved in
MeOH (5 mL) and water (5 mL). NaOH (26 mg, 0.66 mmol, 3 equiv.) was
added and solution was stirred for 3 h at 100.degree. C. The
reaction mixture was extracted with ethyl acetate. Subsequently,
the aqueous layer was neutralized to pH=2 and was extracted with
ethyl acetate. The organic extracts of acidic aqueous solution were
combined and concentrated on a rotary evaporator. The remaining
residue was freeze-dried to yield the title compound ZBB27 in 80
mg. ESI-MS calculated for
C.sub.27H.sub.22N.sub.5O.sub.4[M+H].sup.+=480.16, Obtained: 480.33.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.26 (dd, J=4.5, 1.6 Hz, 1H),
9.05 (d, J=7.6 Hz, 1H), 8.59 (dd, J=8.7, 1.6 Hz, 1H), 8.38 (d,
J=7.6 Hz, 1H), 7.86 (dd, J=8.7, 4.5 Hz, 1H), 7.56 (s, 1H), 6.39 (s,
1H), 3.33 (s, 3H), 3.02 (s, 3H), 2.29 (s, 3H), 2.10 (s, 3H).
##STR00573##
Cpd. No. 331 salt was prepared from amide condensation of ZBB27 and
1-methyl-4-piperidinamine using EDCI-HOBT condition. 75% yield.
ESI-MS calculated for
C.sub.33H.sub.34N.sub.7O.sub.3[M+H].sup.+=576.27, Obtained: 576.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.18 (d, J=2.8 Hz, 1H), 8.92
(d, J=7.6 Hz, 1H), 8.38 (dd, J=8.6, 1.5 Hz, 1H), 8.26 (d, J=7.6 Hz,
1H), 7.70 (dd, J=8.6, 4.3 Hz, 1H), 7.55 (s, 1H), 6.32 (s, 1H),
4.45-4.28 (m, 1H), 3.75-3.55 (m, 2H), 3.45-3.15 (m, 5H), 3.00 (s,
3H), 2.97 (s, 3H), 2.56-1.96 (m, 10H).
##STR00574##
Cpd. No. 332 salt was prepared from amide condensation of ZBB27 and
1-(tetrahydro-2H-pyran-4-yl)-4-piperidinamine using EDCI-HOBT
condition. 80% yield. ESI-MS calculated for
C.sub.37H.sub.40N.sub.7O.sub.4[M+H].sup.+=646.31, Obtained:
646.44.
##STR00575##
Cpd. No. 333 salt was prepared from amide condensation of ZBB27 and
1-(oxetan-3-yl)piperidin-4-amine using EDCI-HOBT condition. 80%
yield. ESI-MS calculated for
C.sub.35H.sub.36N.sub.7O.sub.4[M+H].sup.+=618.28, Obtained:
618.33.
##STR00576##
Cpd. No. 334 salt was prepared from amide condensation of ZBB27 and
1-methylpiperazine using EDCI-HOBT condition. 80% yield. ESI-MS
calculated for C.sub.32H.sub.32N.sub.7O.sub.3[M+H].sup.+=562.25,
Obtained: 562.33. .sup.1H NMR (300 MHz, MeOD) .delta. 9.14 (d,
J=3.1 Hz, 1H), 8.31 (d, J=8.3 Hz, 1H), 8.25-8.13 (m, 2H), 7.67 (dd,
J=8.6, 4.2 Hz, 1H), 7.54 (s, 1H), 6.34 (s, 1H), 3.92-3.31 (m, 8H),
3.30 (s, 3H), 3.02 (s, 3H), 3.01 (s, 3H), 2.29 (s, 3H), 2.10 (s,
3H).
##STR00577##
Cpd. No. 335 salt was prepared from amide condensation of ZBB27 and
4-aminotetrahydropyran using EDCI-HOBT condition. 70% yield. ESI-MS
calculated for C.sub.32H.sub.31N.sub.6O.sub.4[M+H].sup.+=563.24,
Obtained: 563.33. .sup.1H NMR (300 MHz, MeOD) .delta. 9.22 (dd,
J=4.2, 1.7 Hz, 1H), 8.96 (d, J=7.6 Hz, 1H), 8.39 (dd, J=8.7, 1.7
Hz, 1H), 8.28 (d, J=7.6 Hz, 1H), 7.72 (dd, J=8.6, 4.3 Hz, 1H), 7.57
(s, 1H), 6.33 (s, 1H), 4.43-4.30 (m, 1H), 4.13-3.98 (m, 2H),
3.76-3.60 (m, 2H), 3.29 (s, 3H), 3.02 (s, 3H), 2.29 (s, 3H),
2.22-2.04 (m, 5H), 1.94-1.74 (m, 2H).
##STR00578##
Cpd. No. 336 salt was prepared from amide condensation of ZBB27 and
3-oxetanamine using EDCI-HOBT condition. 80% yield. ESI-MS
calculated for C.sub.30H.sub.27N.sub.6O.sub.4[M+H].sup.+=535.20,
Obtained: 535.33.
##STR00579##
1-Bromonaphthalene (2.8 g), Pd(OAc).sub.2 (0.6 g), P(tBu).sub.3
(0.5 g) and tBuONa (2 g) were added to a round-bottom flask
Anhydrous PhMe (60 mL) was added via a syringe and the flask was
degassed and refilled with nitrogen. 1-Boc-piperazine (4 g) was
added and the system was degassed again followed by heating at
60.degree. C. for 16 h. The reaction mixture was cooled to ambient
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound ZBB65 was isolated in 3.5
g. ESI-MS calculated for
C.sub.19H.sub.25N.sub.2O.sub.2[M+H].sup.+=313.19, Obtained:
313.24.
##STR00580##
ZBB65 (4.3 g) was dissolved in CH.sub.3CN (80 mL). NBS (2.7 g) was
added in small portions and the mixture was stirred at room
temperature for 10 h. The volatile components were removed on a
rotary evaporator. The remaining residue was purified by flash
column chromatography to yield ZBB66 in 3.5 g. ESI-MS calculated
for C.sub.19H.sub.24BrN.sub.2O.sub.2 [M+H].sup.+=391.10, Obtained:
391.22.
##STR00581##
ZBB66 (1.6 g, 4.16 mmol), bis(pinacolato)diboron (2.13 g, 8.4 mmol,
2.0 equiv.), and potassium acetate (1.6 g, 16 mmol, 4.0 equiv.)
were added to a round-bottom flask. Anhydrous 1,4-dixoane (20 mL)
was added via a syringe and the flask was degassed and refilled
with nitrogen. Pd(dppf)Cl.sub.2 (322 mg, 0.46 mmol, 0.1 equiv.) was
added and the system was degassed again followed by heating at
100.degree. C. for 16 h. The reaction mixture was cooled to ambient
temperature and diluted by CH.sub.2Cl.sub.2. The solution was
filtered through a pad of celite and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography. The title compound ZBB73 was isolated in 1.3
g. ESI-MS calculated for
C.sub.25H.sub.36BN.sub.2O.sub.4[M+H].sup.+=439.27, Obtained:
439.33.
##STR00582##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 5.4 g, 16 mmol, 1.0 equiv.) and ZBB73 (16.75 g, 37
mmol, 2.0 equiv.), 1,2-dimethoxyethane (150 mL), and
Na.sub.2CO.sub.3 (2 M, 50 mL) were added. The system was degassed
to remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (1.3 g, 1.6 mmol, 0.1 equiv.)
was added and the system was degassed and refilled with nitrogen.
The reaction mixture was heated at reflux for 16 h. The reaction
was quenched with water and the aqueous layer was extracted with
ethyl acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
dissolved in CH.sub.2Cl.sub.2 (50 mL) and TFA (50 mL). The reaction
mixture was stirred for 3 hours at room temperature. The volatile
components were removed on a rotary evaporator and the residue was
purified by flash column chromatography to yield the title compound
Cpd. No. 337 in 2.0 g. ESI-MS calculated for
C.sub.31H.sub.31N.sub.6O.sub.2[M+H].sup.+=519.25, Obtained: 519.33.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.52-8.47 (m, 1H), 8.01 (d,
J=7.8 Hz, 1H), 7.81-7.72 (m, 2H), 7.64-7.52 (m, 3H), 6.20 (s, 1H),
3.68-3.50 (m, 8H), 3.17 (s, 3H), 3.02 (s, 3H), 2.26 (s, 3H), 2.07
(s, 3H).
##STR00583##
The Cpd. No. 337 (20 mg), formaldehyde (0.2 mL, 37% in H.sub.2O)
and NaBH(OAc).sub.3 (65 mg) was dissolved in ClCH.sub.2CH.sub.2Cl
(10 mL) and the mixture was stirred overnight. Then water and Ethyl
acetate was slowly added. The aqueous layer was extracted with
EtOAc. The combined EtOAc extracts were washed with H.sub.2O, dried
over Na.sub.2SO.sub.4, and concentrated under reduced pressure to
afford Cpd. No. 338 (8 mg) after HPLC purification. ESI-MS
calculated for C.sub.32H.sub.33N.sub.6O.sub.2[M+H].sup.+=533.26,
Obtained: 533.34.
##STR00584##
The Cpd. No. 337 (20 mg), and NaBH(OAc).sub.3 (65 mg) was dissolved
in acetone (1 mL) and the mixture was stirred overnight. Then water
and Ethyl acetate was slowly added. The aqueous layer was extracted
with EtOAc. The combined EtOAc extracts were washed with H.sub.2O,
dried over Na.sub.2SO.sub.4, and concentrated under reduced
pressure to afford Cpd. No. 339 (12 mg) after HPLC purification.
ESI-MS calculated for
C.sub.34H.sub.37N.sub.6O.sub.2[M+H].sup.+=561.29, Obtained: 561.33.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.55-8.51 (m, 1H), 8.02 (d,
J=7.8 Hz, 1H), 7.82-7.74 (m, 2H), 7.68-7.52 (m, 3H), 6.20 (s, 1H),
3.91-3.59 (m, 7H), 3.51-3.35 (m, 2H), 3.18 (s, 3H), 3.03 (s, 3H),
2.27 (s, 3H), 2.08 (s, 3H), 1.54 (d, J=6.7 Hz, 6H).
##STR00585##
The Cpd. No. 337 (20 mg), and acetic anhydride (5 mg) was dissolved
in THF (1 mL) and the mixture was stirred overnight. Then water and
Ethyl acetate was slowly added. The aqueous layer was extracted
with EtOAc. The combined EtOAc extracts were washed with H.sub.2O,
dried over Na.sub.2SO.sub.4, and concentrated under reduced
pressure to afford Cpd. No. 340 (6 mg) after HPLC purification.
ESI-MS calculated for
C.sub.33H.sub.33N.sub.6O.sub.3[M+H].sup.+=561.26, Obtained: 561.34.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.58-8.50 (m, 1H), 7.97 (d,
J=7.8 Hz, 1H), 7.79-7.72 (dd, J=11.1, 4.7 Hz, 2H), 7.65-7.56 (m,
1H), 7.54 (s, 1H), 7.51 (d, J=7.9 Hz, 1H), 6.23 (s, 1H), 4.12-3.86
(m, 4H), 3.36-3.22 (m, 4H), 3.20 (s, 3H), 3.02 (s, 3H), 2.28 (s,
3H), 2.23 (s, 3H), 2.09 (s, 3H).
##STR00586##
Cpd. No. 341-TFA salt was prepared from amide condensation of
ZBA301 and 3-aminooxetane using EDCI-HOBT condition. 50% yield.
ESI-MS calculated for
C.sub.31H.sub.34N.sub.5O.sub.4[M+H].sup.+=540.26, Obtained: 540.33.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.65-8.49 (m, 2H), 8.34 (dt,
J=6.4, 1.7 Hz, 1H), 7.59 (d, J=2.4 Hz, 1H), 7.24 (s, 1H), 4.93-4.57
(m, 3H), 3.95-3.70 (m, 2H), 3.67 (d, J=5.4 Hz, 3H), 3.00 (d, J=2.0
Hz, 3H), 2.33 (s, 3H), 2.15 (s, 3H), 1.52 (d, J=1.3 Hz, 9H).
##STR00587##
ZBA301 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). 3-((tert-butyldimethylsilyl)oxy)-3-methylcyclobutan-1-amine
(40 mg) was added and the reaction mixture was stirred for 12 h.
The reaction was quenched with NaHCO.sub.3 saturated solution and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were concentrated on a rotary evaporator. The
remaining residue was dissolved in TFA (2 mL) and DCM (2 mL). The
mixture was stirred for 3 hours and was concentrated on a rotary
evaporator. The remaining residue was purified by reverse phase
HPLC affording the Cpd. No. 342 as a salt of CF.sub.3CO.sub.2H (9
mg). ESI-MS calculated for
C.sub.33H.sub.35N.sub.5O.sub.4[M+H].sup.+=568.29, Obtained: 568.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.40 (t, J=1.6 Hz, 1H), 8.37
(t, J=1.7 Hz, 1H), 8.22 (t, J=1.7 Hz, 1H), 7.58 (s, 1H), 7.31 (s,
1H), 4.23-4.08 (m, 1H), 3.69 (s, 3H), 3.00 (s, 3H), 2.60-2.45 (m,
2H), 2.33 (s, 3H), 2.23 (td, J=9.0, 2.2 Hz, 2H), 2.15 (s, 3H), 1.53
(s, 9H), 1.42 (s, 3H).
##STR00588##
Cpd. No. 343-TFA salt was prepared from amide condensation of
ZBA301 and piperidin-4-ol using EDCI-HOBT condition. 60% yield.
ESI-MS calculated for
C.sub.33H.sub.38N.sub.5O.sub.4[M+H].sup.+=568.29, Obtained: 568.43.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.14 (t, J=1.7 Hz, 1H),
7.95-7.90 (m, 2H), 7.58 (s, 1H), 7.27 (s, 1H), 4.30-4.15 (m, 1H),
3.99-3.88 (m, 1H), 3.80-3.65 (m, 4H), 3.51-3.30 (m, 2H), 3.00 (s,
3H), 2.33 (s, 3H), 2.15 (s, 3H), 2.06-1.78 (m, 2H), 1.69-1.42 (m,
11H).
##STR00589##
ZBA301 (20 mg, 0.05 mmol), EDCI-HCl (100 mg, 0.5 mmol), and
HOBt-H.sub.2O (70 mg, 0.5 mmol) were added to a round-bottom flask.
EtN(i-Pr).sub.2 (0.1 mL) was added followed by addition of DMF (2.5
mL). (S)-4-Amino-1-(trityloxy)butan-2-ol (40 mg) was added and the
reaction mixture was stirred for 12 h. The reaction was quenched
with NaHCO.sub.3 saturated solution and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
344 as a salt of CF.sub.3CO.sub.2H (10 mg). ESI-MS calculated for
C.sub.32H.sub.38N.sub.5O.sub.5[M+H].sup.+=572.28, Obtained: 572.45.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.39 (t, J=1.6 Hz, 1H), 8.36
(t, J=1.7 Hz, 1H), 8.21 (t, J=1.7 Hz, 1H), 7.58 (s, 1H), 7.30 (s,
1H), 3.80-3.48 (m, 8H), 3.00 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H),
1.99-1.62 (m, 2H), 1.52 (s, 9H).
##STR00590##
Cpd. No. 345-TFA salt was prepared from amide condensation of
ZBA301 and cis-4-Amino-cyclohexanol using EDCI-HOBT condition. 60%
yield. ESI-MS calculated for
C.sub.34H.sub.40N.sub.5O.sub.4[M+H].sup.+=582.30, Obtained: 582.55.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.39 (t, J=1.6 Hz, 1H), 8.36
(t, J=1.7 Hz, 1H), 8.21 (t, J=1.7 Hz, 1H), 7.58 (s, 1H), 7.32 (s,
1H), 4.06-3.90 (m, 2H), 3.70 (s, 3H), 3.00 (s, 3H), 2.33 (s, 3H),
2.15 (s, 3H), 1.97-1.59 (m, 8H), 1.53 (s, 9H).
##STR00591##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 54 mg, 1.0 equiv.) and
3-(tert-butyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(137 mg, 2.0 equiv.), 1,2-dimethoxyethane (15 mL), and
Na.sub.2CO.sub.3 (2 M, 5 mL) were added. The system was degassed to
remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (13 mg) was added and the system
was degassed and refilled with nitrogen. The reaction mixture was
heated at reflux for 16 h. The reaction was quenched with water and
the aqueous layer was extracted with ethyl acetate. The organic
layers were combined and the volatile components were removed on a
rotary evaporator. The residue was purified by flash column
chromatography to yield the title compound Cpd. No. 346 in 16 mg.
ESI-MS calculated for
C.sub.26H.sub.28N.sub.5O.sub.2[M+H].sup.+=442.22, Obtained: 442.44.
.sup.1H NMR (300 MHz, MeOD) .delta. 9.13 (s, 2H), 8.69 (d, J=1.8
Hz, 1H), 7.58 (s, 1H), 7.21 (s, 1H), 3.72 (s, 3H), 2.98 (s, 3H),
2.33 (s, 3H), 2.15 (s, 3H), 1.55 (s, 9H).
##STR00592##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 54 mg, 1.0 equiv.) and
4-(tert-butyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(137 mg, 2.0 equiv.), 1,2-dimethoxyethane (15 mL), and
Na.sub.2CO.sub.3 (2 M, 5 mL) were added. The system was degassed to
remove oxygen and nitrogen was refilled.
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (13 mg) was added and the system
was degassed and refilled with nitrogen. The reaction mixture was
heated at reflux for 16 h. The reaction was quenched with water and
the aqueous layer was extracted with ethyl acetate. The organic
layers were combined and the volatile components were removed on a
rotary evaporator. The residue was purified by flash column
chromatography to yield the title compound Cpd. No. 347 in 4 mg.
ESI-MS calculated for
C.sub.26H.sub.28N.sub.5O.sub.2[M+H].sup.+=442.22, Obtained: 442.46.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.98 (d, J=5.4 Hz, 1H), 8.35
(d, J=1.2 Hz, 1H), 7.91 (dd, J=5.3, 1.9 Hz, 1H), 7.82 (s, 1H), 7.55
(s, 1H), 3.81 (s, 3H), 3.00 (s, 3H), 2.35 (s, 3H), 2.17 (s, 3H),
1.51 (s, 9H).
##STR00593##
To a round-bottom flask,
4-(4-chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-
isoxazole (S13, 54 mg, 1.0 equiv.) and
(4-isopropylpyridine-3-yl)boronic acid (100 mg, 2.0 equiv.),
1,2-dimethoxyethane (15 mL), and Na.sub.2CO.sub.3 (2 M, 5 mL) were
added. The system was degassed to remove oxygen and nitrogen was
refilled. Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (13 mg) was added and
the system was degassed and refilled with nitrogen. The reaction
mixture was heated at reflux for 16 h. The reaction was quenched
with water and the aqueous layer was extracted with ethyl acetate.
The organic layers were combined and the volatile components were
removed on a rotary evaporator. The residue was purified by flash
column chromatography to yield the title compound Cpd. No. 348 in
10 mg. ESI-MS calculated for
C.sub.25H.sub.26N.sub.5O.sub.2[M+H].sup.+=428.20, Obtained: 428.45.
.sup.1H NMR (300 MHz, MeOD) .delta. 8.98 (d, J=5.5 Hz, 1H), 8.86
(s, 1H), 8.03 (d, J=5.6 Hz, 1H), 7.57 (s, 1H), 6.60 (s, 1H), 3.59
(s, 3H), 3.11-2.92 (m, 4H), 2.32 (s, 3H), 2.13 (s, 3H), 1.31 (d,
J=6.7 Hz, 3H), 1.22 (d, J=6.6 Hz, 3H).
Synthesis of Cpd No. 350 (TFA salt)
##STR00594##
To a round-bottom flask, 4-Amino-1-Boc-piperidine (2 g) was
dissolved in THF (30 mL) and water (30 mL). NaHCO.sub.3 (8 g) and
CbzCl (1.5 mL) was added and the solution was stirred for 10 h at
rt. The reaction mixture was extracted with ethyl acetate. The
organic extracts were combined and concentrated on a rotary
evaporator. The remaining residue was dissolved in TFA (4 mL) and
DCM (40 mL) and the solution was stirred for 4 h at rt. The
solution was concentrated on a rotary evaporator. Then aq.
NaHCO.sub.3 (30 mL) and the reaction mixture was extracted with
ethyl acetate. The organic extracts were combined and concentrated
on a rotary evaporator to give ZBA240 (1.6 g) which was used
directly in the next step.
##STR00595##
To a round-bottom flask, ZBA240 (2.3 g) was dissolved in DMF (30
mL). K.sub.2CO.sub.3 (2 g), tert-butyl (3-bromopropyl)carbamate
(2.4 g) and NaI (750 mg) was added and the solution was stirred for
2 h at 60.degree. C. The reaction mixture was extracted with ethyl
acetate. The organic layers were combined and the volatile
components were removed on a rotary evaporator. The residue was
purified by flash column chromatography to yield the title compound
ZBA241 in 1.8 g. ESI-MS calculated for
C.sub.21H.sub.34N.sub.3O.sub.4[M+H].sup.+=392.25, Obtained:
392.44.
##STR00596##
To a round-bottom flask, ZBA241 (300 mg), 10% Pd/C (100 mg), MeOH
(20 mL) were added. The system was degassed to remove oxygen and
hydrogen was refilled. the solution was stirred for 10 h at rt. The
solution was filtered through a pad of celite and the volatile
components were removed on a rotary evaporator. The residue
ZBA241-2 was directly used next step without purification.
##STR00597##
Cpd. No. 289 (20 mg), HBTU (24 mg), HOBt-H.sub.2O (6 mg) and DMF (1
mL) were added to a round-bottom flask. EtN(i-Pr).sub.2 (0.05 mL)
was added followed by addition of ZBA241-2 (37 mg) was added and
the reaction mixture was stirred for 12 h. The reaction was
quenched with NaHCO.sub.3 saturated solution and the aqueous layer
was extracted with ethyl acetate. The combined organic layers were
concentrated on a rotary evaporator. The remaining residue was
dissolved in TFA (2 mL) and DCM (2 mL). The mixture was stirred for
3 hours and was concentrated on a rotary evaporator. The remaining
residue was purified by reverse phase HPLC affording the Cpd. No.
349 as a salt of CF.sub.3CO.sub.2H (10 mg). ESI-MS calculated for
C.sub.34H.sub.36FN.sub.8O.sub.3[M+H].sup.+=623.28, Obtained:
623.43.
##STR00598##
To a round-bottom flask, Cpd. No. 349 (19 mg) was dissolved in DMSO
(1 mL). 5-FAM, SE (5-Carboxyfluorescein, Succinimidyl Ester) (43
mg), DIEPS (0.03 mL) was added and the solution was stirred for 2 h
at rt. The mixture was purified by reverse phase HPLC affording the
tracer Cpd. No. 350 as a salt of CF.sub.3CO.sub.2H (6 mg). ESI-MS
calculated for C.sub.55H.sub.46FN.sub.8O.sub.9[M+H].sup.+=981.33,
Obtained: 981.42. .sup.1H NMR (300 MHz, MeOD) .delta. 9.20 (d,
J=4.4 Hz, 1H), 8.51 (s, 1H), 8.36 (dd, J=9.3, 5.4 Hz, 1H), 8.26
(dd, J=8.1, 1.5 Hz, 1H), 7.99 (d, J=4.5 Hz, 1H), 7.82-7.72 (m, 1H),
7.54 (s, 1H), 7.44 (dd, J=9.7, 2.6 Hz, 1H), 7.36 (d, J=7.9 Hz, 1H),
6.75 (d, J=2.2 Hz, 2H), 6.66-6.56 (m, 4H), 6.40 (s, 1H), 4.45-4.25
(m, 1H), 3.80-3.70 (m, 2H), 3.64-3.54 (m, 2H), 3.32-3.16 (m, 4H),
2.45-2.24 (m, 5H), 2.23-1.99 (m, 7H).
##STR00599##
1-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,
5-b]indol-4-yl)quinolin-4(1H)-one (Cpd. No. 351): .sup.1H-NMR (300
MHz, CD.sub.3OD) .delta. ppm 8.51 (dd, J=1.68, 7.77 Hz, 1H), 8.43
(d, J=7.78 Hz, 1H), 7.70-7.54 (m, 2H), 7.43 (s, 1H), 7.32 (dd,
J=0.88, 8.13 Hz, 1H), 6.66 (d, J=7.76 Hz, 1H), 6.26 (s, 1H), 3.32
(s, 3H), 2.89 (s, 3H), 2.26 (s, 3H), 2.08 (s, 3H); ESI-MS m/z
452.50 (M+H).sup.+.
##STR00600##
4-(1-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)-8-methoxy-5H-pyrido[4,3-b]in-
dol-7-yl)-3,5-dimethylisoxazole was prepared as described in
Section 3.2 above. .sup.1HNMR (300 MHz, MeOD-d.sub.4) .delta. 8.60
(d, 1H, J=6.6 Hz), 8.00 (d, 1H, J=6.9 Hz), 7.64-7.66 (m, 6H), 7.11
(s, 1H), 3.78 (s, 3H), 2.35 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H),
2.17 (s, 3H). .sup.13CNMR (300 MHz, MeOD-d.sub.4), .delta. 168.26,
161.16, 155.67, 149.62, 149.10, 142.49, 142.22, 140.20, 137.48,
136.42, 131.02, 130.49, 126.75, 123.83, 122.75, 121.30, 117.06,
114.60, 108.87, 103.95, 56.38, 12.56, 11.74, 11.69, 10.81. ESIMS
m/z [M+H].sup.+ calcd.=464.54. found=464.42.
##STR00601##
4-(1-chloro-8-methoxy-5H-pyrido[4,3-b]indol-7-yl)-3,5-diethylisoxazole
was prepared as described in Section 4.3 above. .sup.1HNMR (300
MHz, DMSO-d.sub.6) .delta. 12.06 (s, 1H), 8.22 (d, 1H, J=5.7 Hz),
7.96 (s, 1H), 7.52 (d, 1H, J=5.7 Hz), 7.49 (s, 1H), 3.89 (s, 3H),
2.65 (q, 2H, J=7.5 Hz), 2.53 (q, 2H, J=7.5 Hz), 1.15 (t, 3H, J=7.5
Hz), 1.04 (t, 3H, J=7.5 Hz).
.sup.13CNMR (300 MHz, DMSO-d.sub.6), .delta. 169.85, 163.67,
152.08, 145.84, 143.62, 143.55, 133.92, 119.93, 119.20, 116.06,
114.30, 111.86, 106.71, 103.19, 55.71, 18.94, 18.49, 11.81. ESIMS
m/z [M+H].sup.+ calcd.=356.67. found=356.83.
To demonstrate the ability of the present BET bromodomain
inhibitors to bind to BET bromodomain proteins, competitive FP
binding assays were designed and performed for recombinant BRD2
BD2, BRD3 BD2, and BRD4 BD2 proteins.
The FAM labeled fluorescent probe (BRD-1F) was synthesized based on
a known small-molecule BET bromodomain inhibitor. K.sub.d values of
BRD-1F to these three proteins were determined by monitoring the
total fluorescence polarization of mixtures composed with the
fluorescent probe at a fixed concentration and proteins with
increasing concentrations up to full saturation. Fluorescence
polarization values were measured using the Infinite M-1000 plate
reader (Tecan U.S., Research Triangle Park, N.C.) in Corning 384
well flat bottom black plates (Corning Life Science). Serial
dilutions of testing protein were mixed with BRD-1F to a final
volume of 80 .mu.l in the assay buffer (100 mM potassium phosphate,
pH 7.5, 100 .mu.g/ml bovine .gamma.-globulin, 0.02% sodium azide,
Invitrogen, with 0.01% Triton X-100 and 2.5% Ethylene Glycol).
Final BRD-1F concentration was 5 nM. Plates were incubated at room
temperature for 1-2 hours with gentle shaking to assure
equilibrium. The polarization values in millipolarization units
(mP) were measured at an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. Equilibrium dissociation constants
(K.sub.d) were then calculated by fitting the sigmoidal
dose-dependent FP increases as a function of protein concentrations
using Graphpad Prism 5.0 software (Graphpad Software, San Diego,
Calif.).
The IC50 and K.sub.i values of compounds were determined in a
competitive binding experiment in which serial dilutions of
compounds competed against fixed concentration of the fluorescent
probe (BRD-1F) for binding to the protein with a fixed
concentration (typically 2 to 3 times the K.sub.d values determined
above) as well. Mixtures of 2 .mu.l of the tested compounds in
Ethylene Glycol and 78 .mu.l of preincubated protein/probe complex
solution in the assay buffer (100 mM potassium phosphate, pH 7.5,
100 .mu.g/ml bovine .gamma.-globulin, 0.02% sodium azide,
Invitrogen with 0.01% Triton X-100) were added into assay plates
and incubated at room temperature for 1 hour with gentle shaking
Final concentrations of proteins were 200 nM, 150 nM and 200 nM in
assays for BRD2 BD2, BRD3 BD2, and BRD4 BD2, respectively. Final
probe concentration is 5 nM in all assays. Negative controls
containing protein/probe complex only (equivalent to 0%
inhibition), and positive controls containing only free probes
(equivalent to 100% inhibition), were included in each assay plate.
FP values were measured as described above. IC.sub.50 values were
determined by nonlinear regression fitting of the competition
curves. The K.sub.i values of competitive inhibitors were
calculated using the derived equation described previously, based
upon the measured IC.sub.50 values, the K.sub.d values of the probe
to different proteins, and the concentrations of the proteins and
probes in the competitive assays.
Table 1 lists binding affinities of several representative
compounds to BRD2 BD2 and BRD4 BD2 proteins.
TABLE-US-00002 TABLE 1 Binding affinities of representative
compounds to BRD2 BD2 and BRD4 BD2 in FP competitive binding
assays. Binding Affinities Cpd. BRD2 BD2 BRD4 BD2 No. ID Structure
IC50 (nM) Ki (nM) IC50 (nM) Ki (nM) Cpd. No. 2 ##STR00602## 286
.+-. 125 42.3 .+-. 32.0 608 .+-. 244 114 .+-. 87 RX-7 ##STR00603##
1350 .+-. 212 366 .+-. 82 2902 850 Cpd. No. 3 ##STR00604## 255 .+-.
114 34.9 .+-. 21.2 514 83.4 Cpd. No. 4 ##STR00605## 349 57.4 873
232 Cpd. No. 17 ##STR00606## 142 .+-. 31 <10 197 .+-. 99 16.4
Cpd. No. 21 ##STR00607## 92.8 .+-. 30.8 <10 310 .+-. 71 37.9
.+-. 10.0 Cpd. No. 34 ##STR00608## 3197 1199 3666 775
Binding affinities of synthesized compounds to BRD2 BD1 and BD2,
BRD3 BD1 and BD2, and BRD4 BD1 and BD2 were also determined by a
label free binding assay using the OctetRED label free biolayer
interferometry (BLI) binding assay.
BLI measures interference pattern changes of light reflected from
an optical layer and a biolayer containing protein targets only or
complexed with interacting partners. The assay principle is similar
to the surface plasmon resonance (SPR) assay in which the target
protein is immobilized on an optical surface and then exposed to
potential binding partners in solution. The interaction between the
binding partner and the immobilized protein changes the optical
properties of the biosensors, resulting in the wavelength shift of
reflecting light which will change the interference pattern.
Association and dissociation rates can be obtained by fitting the
real time wavelength shift of the based on a proper binding model,
from which K.sub.D values can be obtained thereafter.
Biotin labeled BRD proteins (10 .mu.g/ml) in kinetic assay buffer
(PBS, pH 7.4, 0.1% BSA and 0.01% Tween-20) were immobilized on
Super Streptavidin (SSA) sensors for 15 minutes followed by washing
in kinetic buffer for 10 minutes to eliminate any loose
non-specific immobilization. In the same 96-well plate serial
dilutions of testing compounds with concentrations typically
ranging from 0.1-10 times of expected K.sub.d values in the
identical assay buffer were prepared. These protein coated sensors
were then immersed into the testing compound solutions, starting
from the one with the lowest concentration, where compound
association occurs and then returned to the fresh buffer for the
dissociation. The same operation was repeated for the next solution
with higher concentration up to the one with the highest
concentration. Identical procedure was performed again with control
sensors that were immobilized with SAB4 inactive control protein
prepared by following protocols from the manufacturer. Blank buffer
controls were included in both BRD protein sensor and inactive
protein sensor runs. For each kinetic cycle, kinetic curves for
association and dissociation were obtained from raw sensorgrams by
using the double reference subtraction protocol included in the
analysis program (Data Analysis 7.0) provided by the manufacturer,
in which nonspecific interaction and buffer drift were both
corrected. The association and dissociation rate constants
(k.sub.on and k.sub.off) were determined using the global fitting
protocol in the analysis program based on a reversible 1:1 binding
model. The equilibrium association constant (K.sub.A) was
calculated thereafter. All binding data were collected at 30
degree. Assay plates were kept being shaken at 1000 RPM in the
whole experiment time period to avoid mass transport effect.
Table 2 lists the binding affinities of several representative
compounds to BRD2 BD1, BRD2 BD2, BRD3 BD1, BRD3 BD2, BRD4 BD1 and
BRD4 BD2 proteins.
TABLE-US-00003 TABLE 2 Binding affinities of several representative
BET bromodomain inhibitors to BRD2 BD1, BRD2 BD2, BRD3 BD1, BRD3
BD2, BRD4 BD1 and BRD4 BD2 proteins using the biolayer
interferometry (BLI) binding assay. BRD2 BRD3 BRD4 Kd (nM) BD1 BD2
BD1 BD2 BD1 BD2 Cpd. No. 22 34.0 .+-. 2.1 24.4 .+-. 6.2 17.9 .+-.
8.8 18.7 .+-. 4.9 40.0 .+-. 6.4 27.9 .+-. 6.8 Cpd. No. 23 25.6 .+-.
3.7 19.6 .+-. 4.1 17.6 .+-. 2.5 20.7 .+-. 3.6 29.0 .+-. 8.8 12.9
.+-. 2.1 Cpd. No. 25 41.2 .+-. 2.2 35.9 .+-. 8.8 20.7 .+-. 7.8 42.4
.+-. 17.6 60.3 .+-. 17.6 36.3 .+-. 11.2 Cpd. No. 44 33.8 .+-. 8.2
11.0 .+-. 4.7 17.0 .+-. 7.0 10.6 .+-. 1.1 27.0 .+-. 14.5 10.2 .+-.
4.5
Cell growth inhibitory activity of representative BET bromodomain
inhibitors was determined using CellTiter-Glo.RTM. Luminescent Cell
Viability Assay. For leukemia cell lines MV-4-11 (ATCC, Manassas,
Va.) and MOLM-13 (DSMZ, Germany), cells were seeded in 96-well
white opaque cell culture plates at a density of 10,000 cells/well
with serially diluted compounds and incubated at 37.degree. C. in
an atmosphere of 95% air and 5% CO2 for 4 days. Cell viability was
determined using the CellTiter-Glo.RTM. Luminescent Cell Viability
Assay Kit (Promega, Madison, Wis.) according to the manufacture's
instruction. Briefly, a volume of CellTiter-Glo.RTM. Reagent equal
to the volume of cell culture medium was added to each well, and
then the plates were incubated at room temperature for 10-20
minutes. The luminescent signal was measured using a Tecan Infinite
M1000 multimode microplate reader (Tecan, Morrisville, N.C.). The
half maximal inhibitory concentration (IC50) was calculated using
the GraphPad Prism 5 software (GraphPad Software, La Jolla,
Calif.).
For breast cancer cell lines, cells were seeded in 96-well cell
culture plates at a density of 5,000-10,000 cells/well with
serially diluted compounds and incubated at 37.degree. C. in an
atmosphere of 95% air and 5% CO2 for 4 days. All the breast cancer
cell lines were obtained from the ATCC. Cell viability was
determined using the WST-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-t-
etrazolium, monosodium salt) based Cell Counting-8 Kit (Dojindo
Molecular Technologies, Inc., Rockville, Md.) according to the
manufacture's instruction. Briefly, WST-8 was added to each well at
a final concentration of 10% (v/v), and then the plates were
incubated at 37.degree. C. for 1-2 hours for color development. The
absorbance was measured at 450 nm using a SPECTRAmax PLUS plate
reader (Molecular Devices, Sunnyvale, Calif.). The IC50 was
calculated using the GraphPad Prism 5 software.
Table 3 lists the IC50 values for several representative BET
bromodomain inhibitors in the present invention in inhibition of
cell growth in leukemia cell lines. Table 4 lists cell growth
inhibition of compound Cpd. No. 68 in breast cancer cell lines.
TABLE-US-00004 TABLE 3 Cell growth inhibition of several
representative compounds in acute leukemia cell lines. IC50 (nM)
Compounds MV-4-11 Molm-13 Cpd. No. 17 <100 <500 Cpd. No. 21
<100 <500 Cpd. No. 38 <100 <500 Cpd. No. 44 <100
<100 Cpd. No. 68 <100 <100
TABLE-US-00005 TABLE 4 Cell growth inhibition of compound Cpd. No.
68 in breast cancer cell lines. Cell Lines IC50 (nM) BT-474 <300
MDA-MB-157 <300 MDA-MB-231 <300 MDA-MB-436 <300 SK-BR-3
<300
Fluorescence Polarization (FP) Competitive Binding Assays Using
Cpd. No. 350
Fluorescence Polarization (FP) competitive binding studies (see
above) were carried out using the FAM labeled fluorescent probe
Cpd. No. 350 to determine binding affinities of representative
compounds to both BD1 and BD2 of BRD2, BRD3, and BRD4 proteins.
Equilibrium dissociation constants (Id) values of Cpd. No. 350 to
these six proteins were determined from protein saturation
experiments by monitoring the total fluorescence polarization of
mixtures composed with the fluorescent probe at a fixed
concentration and proteins with increasing concentrations up to
full saturation. Serial dilutions of testing protein were mixed
with Cpd. No. 350 to a final volume of 200 .mu.l in the assay
buffer. In order to achieve large dynamic rages, particularly for
BD1 bromodomains, 100 mM phosphate buffer (pH=6.5, 0.01% Triton
X-100 (Sigma, 282103) being added right before assays) was used as
the assay buffer. Final Cpd. No. 350 concentration was 1.5 nM for
all proteins. Plates were incubated at room temperature for 30
minutes with gentle shaking to assure equilibrium. FP values in
millipolarization units (mP) were measured using the Infinite
M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in
Microfluor 1 96-well, black, round-bottom plates (Thermo
Scientific, Waltham, Mass.) at an excitation wavelength of 485 nm
and an emission wavelength of 530 nm. K.sub.d values of Cpd. No.
350, which were calculated by fitting the sigmoidal dose-dependent
FP increases as a function of protein concentrations using Graphpad
Prism 6.0 software (Graphpad Software, San Diego, Calif.), are 2.0,
2.2, 6.5, 0.6, 5.5, and 3.0 nM to BRD2 BD1 and 2, BRD3 BD1 and 2,
and BDR4 BD1 and 2, respectively.
The IC.sub.50 and K.sub.i values of compounds were determined in a
competitive binding experiment as described above. Mixtures of 10
.mu.l of the tested compounds in assay buffer with 40% Ethylene
Glycol and 190 .mu.l of preincubated protein/probe complex solution
in the assay buffer (100 mM potassium phosphate, pH 6.5, 0.01%
Triton X-100) were added into assay plates which were incubated at
room temperature for 30 minutes with gentle shaking Final
concentrations of proteins were 3, 6, 15, 2, 10, and 6 nM in assays
for BD1 and BD2 of BRD2, BRD3, and BRD4 BD2, respectively. Final
probe concentration is 1.5 nM in all assays. Negative controls
containing protein/probe complex only (equivalent to 0%
inhibition), and positive controls containing only free probes
(equivalent to 100% inhibition), were included in each assay plate.
FP values were measured as described above. IC.sub.50 values were
determined by nonlinear regression fitting of the competition
curves. Instead of being calculated from IC.sub.50 values as
described before, K.sub.i values of competitive inhibitors were
obtained directly by nonlinear regression fitting as well, based
upon the K.sub.d values of the probe to different proteins, and
concentrations of the proteins and probes in the competitive assays
(Wang, FEBS Lett. 360; 111 (1995); Zhang et al., Analytical
Biochemistry, 331;138 (2004)).
TABLE-US-00006 TABLE 5 Binding affinities of representative
compounds to recombinant BD1 and BD2 domain proteins of BDR2, BRD3
and BRD4 in fluorescence-polarization based assays using Cpd. No.
350 as the probe BRD2 BRD3 BRD4 Compound BD1 BD2 BD1 BD2 BD1 BD2 ID
K.sub.i (nM) K.sub.i (nM) K.sub.i (nM) K.sub.i (nM) K.sub.i (nM)
K.sub.i (nM) RX-3 215 .+-. 34 109 .+-. 10 144 .+-. 17 63.8 .+-. 6.0
305 .+-. 26 194 .+-. 24 RX-7 650 .+-. 125 498 .+-. 11 730 .+-. 146
241 .+-. 6 1644 .+-. 71 824 .+-. 25 Cpd. No. 1 31.6 .+-. 8.2 34.5
.+-. 2.1 14.7 .+-. 1.0 14.3 .+-. 0.5 47.8 .+-. 1.0 70.1 .+-. 2.0
Cpd. No. 2 138 .+-. 8 89.7 .+-. 6.4 151 .+-. 20 48.7 .+-. 3.3 247
.+-. 29 201 .+-. 5 Cpd. No. 21 48.3 .+-. 1.3 57.7 .+-. 8.1 31.9
.+-. 4.0 25.9 .+-. 1.8 98.8 .+-. 11.6 100 .+-. 16 Cpd. No. 24 58.4
.+-. 1 92.1 .+-. 3.3 38.2 .+-. 1 50.9 .+-. 5.8 116 .+-. 5 134 .+-.
42 Cpd. No. 352 62.6 .+-. 9.0 52.9 .+-. 2.7 31.8 .+-. 1.5 35.0 .+-.
2.0 103 .+-. 3 98.1 .+-. 6.1 Cpd. No. 22 21.0 .+-. 3.3 15.4 .+-.
3.2 12.9 .+-. 2.9 4.2 .+-. 0.4 44.1 .+-. 6.4 16.1 .+-. 2.8 Cpd. No.
23 11.1 .+-. 1.0 11.7 .+-. 3.0 7.3 .+-. 0.1 3.2 .+-. 0.5 24.7 .+-.
1.0 12.2 .+-. 1.6 Cpd. No. 25 12.2 .+-. 1.7 22.2 .+-. 2.8 10.4 .+-.
1.0 9.4 .+-. 1.0 26.9 .+-. 1.0 38.0 .+-. 2.2 RX-38 760 .+-. 240
1884 .+-. 432 703 .+-. 432 1279 .+-. 1069 2814 .+-. 782 2182 .+-.
132 RX-39 1716 .+-. 892 638 .+-. 70 668 .+-. 82 406 .+-. 192 1243
.+-. 549 478 .+-. 69 Cpd. No. 33 1668 .+-. 448 909 .+-. 272 1219
.+-. 100 348 .+-. 12 1726 .+-. 17 867 .+-. 107 RX-27 5452 .+-. 1916
2837 .+-. 574 5029 .+-. 1014 2047 .+-. 142 4842 .+-. 29 1948 .+-.
175 RX-45 3438 .+-. 1985 >10000 8322 .+-. 3220 Cpd. No. 68 3.2
2.7 5.1 0.65 7.3 1.7 Cpd. No. 73 15.5 8.7 10.2 2.6 35.3 7.8 Cpd.
No. 183 5.2 8.8 6.1 4.7 7.9 11.7 Cpd. No. 196 4.3 3.5 9.7 1.4 12.3
7.0 Cpd. No. 197 6.2 3.6 10.5 1.3 17.1 8.9 Cpd. No. 207 5.3 5.1
10.0 1.3 14.7 4.1 Cpd. No. 211 5.7 4.9 10.8 1.2 17.2 5.2 Cpd. No.
212 5.7 4.6 10.2 1.0 17.0 4.5 Cpd. No. 213 10.5 7.1 14.0 2.2 20.8
6.5 Cpd. No. 319 2.2 5.2 6.3 1.1 9.0 5.3 Cpd. No. 322 3.2 7.4 7.7
2.3 10.2 7.4 Cpd. No. 316 4.6 2.6 7.5 0.82 11.3 2.9 Cpd. No. 317
16.7 7.8 22.9 3.5 38.6 7.1
Cell Viability Assays
The effect of representative BET bromodomain inhibitors on cell
viability was determined in a 4-day proliferation assay. Cells were
maintained in the appropriate culture medium with 10% FBS at
37.degree. C. and an atmosphere of 5% CO2. All the cell lines were
used within three months of thawing fresh vials.
Cells were seeded in 96-well flat bottom (Corning COSTAR, Corning,
N.Y., cat#3595) or white opaque cell culture plates (BD Falcon,
cat#353296) at a density of 3,000-10,000 cells/well in 75 .mu.l of
culture medium. Compounds were serially diluted in the appropriate
medium, and 75 .mu.l of the diluted compounds were added to the
appropriate wells of the cell plate. After the addition of
compounds, the cells were incubated at 37.degree. C. in an
atmosphere of 5% CO2 for 4 days. Cell viability was determined
using the CellTiter-Glo.RTM. Luminescent Cell Viability Assay Kit
(Promega, Madison, Wis.) for MOLM-13 cells and WST
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-t-
etrazolium, monosodium salt) Cell Counting-8 Kit (Dojindo Molecular
Technologies, Inc., Rockville, Md.) for MDA-MB-436 cells according
to the manufacturers' instructions.
For the WST assay, WST-8 reagent was added to each well at a final
concentration of 10% (v/v), and then the plates were incubated at
37.degree. C. for 1-2 hours for color development. The absorbance
was measured at 450 nm using a SPECTRAmax PLUS plate reader
(Molecular Devices, Sunnyvale, Calif.). The readings were
normalized to the DMSO-treated cells and the half maximal
inhibitory concentration (IC50) was calculated by nonlinear
regression (four parameters sigmoid fitted with variable slope,
least squares fit, and no constraint) analysis using the GraphPad
Prism 5 software (GraphPad Software, La Jolla, Calif.).
For the CellTiter-Glo assay, 100 .mu.l of CellTiter-Glo.RTM.
Reagent was added to each well, and then the plates were incubated
at room temperature for 10-20 minutes. The luminescent signal was
measured using a Tecan Infinite M1000 multimode microplate reader
(Tecan, Morrisville, N.C.). The readings were normalized to the
DMSO-treated cells and the IC50 was calculated by nonlinear
regression (four parameters sigmoid fitted with variable slope,
least squares fit, and no constraint) analysis using the GraphPad
Prism 5 software.
TABLE-US-00007 TABLE 6 Inhibition of cell growth by representative
compounds in leukemia MOLM-13 and breast cancer MDA-MB-436 cell
lines. Cell Growth Inhibition (IC.sub.50 (nM)) Compound ID. MOLM-13
Cell Line MDA-436 Cell Line No. (CellTiter-Glo assay) (WST assay)
Cpd. No. 1 550 .+-. 303 Cpd. No. 2 1042 .+-. 158 Cpd. No. 21 311
.+-. 12 Cpd. No. 24 280 .+-. 35 Cpd. No. 22 183 .+-. 38 Cpd. No. 23
104 .+-. 16 Cpd. No. 25 343 .+-. 5 119.0 Cpd. No. 26 431.1 696.5
Cpd. No. 352 751.3 723.9 Cpd. No. 183 20 93 Cpd. No. 196 6.8 53
Cpd. No. 197 17.3 101 Cpd. No. 207 5.3 58.1 Cpd. No. 211 10.4 140
Cpd. No. 212 22 100 Cpd. No. 213 48 225 Cpd. No. 319 10 67.4 Cpd.
No. 322 19.4 63.9 Cpd. No. 316 7.4 45.2 Cpd. No. 317 31.6 169.1
TABLE-US-00008 TABLE 7 Cell growth inhibition of representative
compounds in the prostate VCaP cell line (cells were treated with
drug for 4 days and cell viability was measured by Cell TiterGLO
assay). VCaP cells Compound ID (IC.sub.50 (nM)) JQ1 48 I-BET762 500
I-BET151 862 Cpd. No. 23 117 Cpd. No. 68 148 Cpd. No. 73 156 Cpd.
No. 90 20 Cpd. No. 101 25
TABLE-US-00009 TABLE 8 Cell growth inhibition of representative
compounds in the leukemia MV4; 11, AML-2 and K562 cell lines. MV4;
11 AML-2 K562 Cpd. ID No. (IC.sub.50(nM)) (IC.sub.50(nM))
(IC.sub.50(nM)) Cpd. No. 1 178 .+-. 114 148 .+-. 64 >2000 Cpd.
No. 2 1074 .+-. 195 217 .+-. 61 >2000 Cpd. No. 21 124 .+-. 39
216 .+-. 43 >2000 Cpd. No. 24 83 .+-. 41 173 .+-. 89 >2000
Cpd. No. 22 61 .+-. 28 101 .+-. 22 >2000 Cpd. No. 23 17 .+-. 3
104 .+-. 5 >2000 Cpd. No. 25 65 .+-. 24 163 .+-. 2 >2000
* * * * *
References